Bitraf - Brukerbidrag [nb]

Biolaboratoriet

2016-09-19T17:47:50Z

Jarlemag: /* Avholdte meetups */

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

*Monday September 5 2016: Bioraf byggekveld: http://www.meetup.com/bitraf/events/233816161/ We placed a book shelf in the main working space and put the equipment on it for easier access.

*Monday September 19 2016: Sequence Alignment Workshop: https://www.meetup.com/bitraf/events/234064840/

=Planlagte meetups=

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

https://www.theconsumablescompany.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

https://github.com/biohackacademy

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-09-07T22:36:58Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

*Monday September 5 2016: Bioraf byggekveld: http://www.meetup.com/bitraf/events/233816161/ We placed a book shelf in the main working space and put the equipment on it for easier access.

=Planlagte meetups=

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

https://www.theconsumablescompany.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

https://github.com/biohackacademy

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-09-03T09:27:03Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

=Planlagte meetups=

Monday September 5 2016: Bioraf byggekveld: http://www.meetup.com/bitraf/events/233816161/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

https://www.theconsumablescompany.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

https://github.com/biohackacademy

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-09-01T22:27:22Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

=Planlagte meetups=

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

https://www.theconsumablescompany.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

https://github.com/biohackacademy

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-08-27T17:25:35Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

=Planlagte meetups=

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

https://www.theconsumablescompany.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-08-25T21:05:11Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

=Planlagte meetups=

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

BioHackerLab/Consumables

2016-08-16T22:15:40Z

Jarlemag: Ny side: Notes on reagents and other consumables. =Growth media= *LB *YPD. See http://www.sigmaaldrich.com/technical-documents/articles/biology/Introduction-yeast-media.html

Notes on reagents and other consumables.

=Growth media=

*LB
*YPD. See http://www.sigmaaldrich.com/technical-documents/articles/biology/Introduction-yeast-media.html

Biolaboratoriet

2016-08-16T20:37:35Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

=Planlagte meetups=

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x lavhastighets mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

BioHackerLab/Equipment

2016-08-15T17:34:29Z

Jarlemag:

An overview of available equipment and equipment of interest. See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Liquid handling=

http://www.pipettecalibration.net/pipette_calibration_full.html

http://www.pipettecalibration.net/pipette-calibration-files/guide-to-pipetting-2.pdf

http://www.ttelaboratories.com/TTE-University/ISO-8655

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf
*https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

Kalibreringssett Kern F1: https://www.vektekspert.no/kalibreringssett-kern-f1-323-324.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

Calibration weight sizes for user tests: According to Good Weighing Practice (GWP) recommendations by Mettler Toledo, two weights should be used for user tests (https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf):
*Weight 1: Largest OIML or ASTM nomination which equals or is smaller than the capacity of the balance or scale
*Largest OIML or ASTM nomination which equals or is smaller than 5% of the capacity of the balance or scale.

Thus for a 100 g capacity scale, a 100g weight and a 5 g weight should be used.

See also USP General Chapter 41: "A test weight is suitable if it has a mass
between 5% and 100% of the balance's capacity."

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-15T16:08:49Z

Jarlemag:

An overview of available equipment and equipment of interest. See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Liquid handling=

http://www.pipettecalibration.net/pipette_calibration_full.html

http://www.pipettecalibration.net/pipette-calibration-files/guide-to-pipetting-2.pdf

http://www.ttelaboratories.com/TTE-University/ISO-8655

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf
*https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

Calibration weight sizes for user tests: According to Good Weighing Practice (GWP) recommendations by Mettler Toledo, two weights should be used for user tests (https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf):
*Weight 1: Largest OIML or ASTM nomination which equals or is smaller than the capacity of the balance or scale
*Largest OIML or ASTM nomination which equals or is smaller than 5% of the capacity of the balance or scale.

Thus for a 100 g capacity scale, a 100g weight and a 5 g weight should be used.

See also USP General Chapter 41: "A test weight is suitable if it has a mass
between 5% and 100% of the balance's capacity."

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-15T10:53:33Z

Jarlemag:

An overview of available equipment and equipment of interest.

=Liquid handling=

http://www.pipettecalibration.net/pipette_calibration_full.html

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf
*https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

Calibration weight sizes for user tests: According to Good Weighing Practice (GWP) recommendations by Mettler Toledo, two weights should be used for user tests (https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf):
*Weight 1: Largest OIML or ASTM nomination which equals or is smaller than the capacity of the balance or scale
*Largest OIML or ASTM nomination which equals or is smaller than 5% of the capacity of the balance or scale.

Thus for a 100 g capacity scale, a 100g weight and a 5 g weight should be used.

See also USP General Chapter 41: "A test weight is suitable if it has a mass
between 5% and 100% of the balance's capacity."

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-15T00:00:47Z

Jarlemag: /* Background reading */

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf
*https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

Calibration weight sizes for user tests: According to Good Weighing Practice (GWP) recommendations by Mettler Toledo, two weights should be used for user tests (https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf):
*Weight 1: Largest OIML or ASTM nomination which equals or is smaller than the capacity of the balance or scale
*Largest OIML or ASTM nomination which equals or is smaller than 5% of the capacity of the balance or scale.

Thus for a 100 g capacity scale, a 100g weight and a 5 g weight should be used.

See also USP General Chapter 41: "A test weight is suitable if it has a mass
between 5% and 100% of the balance's capacity."

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:57:11Z

Jarlemag: /* Calibration */

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

Calibration weight sizes for user tests: According to Good Weighing Practice (GWP) recommendations by Mettler Toledo, two weights should be used for user tests (https://www.ncsli.org/c/f/p13/REG_2013.PRE.1167.1908.pdf):
*Weight 1: Largest OIML or ASTM nomination which equals or is smaller than the capacity of the balance or scale
*Largest OIML or ASTM nomination which equals or is smaller than 5% of the capacity of the balance or scale.

Thus for a 100 g capacity scale, a 100g weight and a 5 g weight should be used.

See also USP General Chapter 41: "A test weight is suitable if it has a mass
between 5% and 100% of the balance's capacity."

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:50:02Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML R 111-1, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument." According to OIML R 76-1, the maximum permissible error for a class I instrument is ± 0.5 e for loads 0 ≤ m ≤ 50 000e, where e is the verification scale interval for the instrument.

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:45:54Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. F1 weights are intended for use in the verification or calibration of class F2 weights and for use with weighing instruments of special accuracy class I and high accuracy class II. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:44:55Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kern OIML F1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-f1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:40:56Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html
*Satisfies requirements for Class II (High) or Class I (special) accuracy according to OIML R076-1.

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:39:30Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

M1 weights are intended for use in the verification or calibration of class M2 weights, and for use with weighing instruments of medium accuracy class III. (OIML R111-1, chapter 1.3)

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:19:02Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

International recommendation OIML R111-1: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:18:20Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

Kalibreringssett Kern M1: https://www.vektekspert.no/kalibreringssett-kern-m1-343-344.html

OIML standard: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T23:17:11Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

Kern OIML M1 calibration weights: https://www.vektekspert.no/kalibreringslodder/klasse-oiml-m1.html

OIML standard: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:55:51Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

OIML standard: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

'''Kern EMB 100-3.''' (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:55:30Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

==Background reading==

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==Calibration==

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

OIML standard: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:54:05Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

Calibration:

Kern OIML M2 calibration weights: https://www.vektekspert.no/kalibreringslodd-kern-m2-357.html

OIML standard: https://www.oiml.org/en/files/pdf_r/r111-1-e04.pdf

INTERNATIONAL
RECOMMENDATION OIML R 76-1 Non-automatic weighing instruments: https://www.oiml.org/en/files/pdf_r/r076-1-e06.pdf

According to OIML, "The error in a weight used for the verification of a weighing instrument shall not exceed 1/3 of the maximum permissible error for an instrument."

For a 1g M2 calibration weight, the permissible error is 3 mg. For a 1g M1 calibration weight, the permissible error is 1 mg.

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:18:01Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/
*http://us.mt.com/dam/MT-NA/WeighMatter/Weighing_the_Right_Way_Brochure.pdf

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:09:44Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/
*https://blog.intelligentwt.com/2014/03/24/what-is-the-minimum-load-of-a-balance/

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T15:08:26Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/
*https://blog.intelligentwt.com/2014/01/22/what-does-linearity-mean-in-a-balance-or-scale/

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T14:52:29Z

Jarlemag:

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. (2761 NOK inkl MVA): https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T14:47:46Z

Jarlemag: /* Weighing */

An overview of available equipment and equipment of interest.

See also: http://www.mlo-online.com/the-quest-for-the-500-home-molecular-biology-laboratory.php

=Thermocyclers=

http://www.the-scientist.com/?articles.view/articleNo/18812/title/How-Low-Can-You-Go---Nineteen-Thermal-Cyclers-Priced-Under--5000/

=DNA quantification=

==GeneQuant/Genequant II==

GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf

*Light path height = 15 mm

==Eppendorf biophotometer 6131==

Manual: http://arboretum.harvard.edu/wp-content/uploads/Biophotometer-manual.pdf

*Device: Single-beam filter photometer with reference beam and fixed wavelengths.

Cuvette types (min volume):
*10 mm macro (1000 uL)
*10 mm semi-micro (400 uL)
*10 mm suction (300 uL)
*10 mm ultra-micro (70 uL)

Wavelengths: 230,260,280,320,562,595

*Photometric random error: <= 0.005A at 1A.
*Photometric systemic error: +- 1% at 1A.

Height of light beams in cuvette: 8.5 mm

Kompatible kuvetter:

https://www.brandtech.com/product/uv-cuvettes/

See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/

===Biophotometer test #1===
Performed August 7 2016. All measurments were performed with previously unused Brand "UV-cuvette micro" cuvettes. For these cuvettes, the specified minimum sample volume is 70 uL.

The oligo program was selected. Programmed factor: 1A260 = 30.0 ug/mL.

The program was blanked with 100 uL nuclease-free water (Dongsheng biotech)

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 single-strand DNA oligomer supplied by Macrogen Inc with expected concentration 10 uM [Note 1] was mixed with 50 uL DSBio NF water to give a solution with expected concentration 5 uM. Somewhat less than 100 uL was measured as sample. Result: 0.0498 ug/uL ~0.05 ug/uL (50 ng/uL) The readings for all wavelengths were as follows: 1.343 A230, 1.661 A260, 0.945 A280, 0.027 A320.

The sample was removed from the photometer, then later replaced and remeasured once with the following result (measurement #5): 0.0492 ug/uL (1.320 A230, 1.639 A260, 0.930 A280, 0.010 A320)

Without being removed from the photometer, the sample was then remeasured thrice in quick succession with the following results [ug/uL]:

*Measurement #6: 0.0494
*Measurement #7: 0.0493
*Measurement #8: 0.0493

The sample was then removed from the photometer, placed back into the photometer and remeasured. This was repeated twice. The results were as follows [ug/uL]:

*Measurement #9: 0.0496
*Measurement #10: 0.0498
*Measurement 11: 0.0502

The cuvette was then measured in one orientation, rotated 180 degress and the sample remeasured. This was repeated once, for a total of four measurements, the results being as follows [ug/uL]:

*Measurement #12: 0.0496 (Original orientation. Logo on cuvette towards front of instrument. Arrow on cuvette towards back of instrument)
*Measurement #13: 0.0509 (Reversed)
*Measurement #14: 0.0506 (Original orientation)
*Measurement #15: 0.0499 (Reversed)

20 uL of a solution of ITS4 primer with expected concentration 10 uM [Note 2] was mixed with 80 uL DSBio NF water. The sample was measured twice, using the dilution correction feature for the second measurement. The results were as follows.

*First measurment: 15.4 ng/uL. A260/A280 = 1.39. Dilution specified: N/A
*Second measurement: 77.3 ng/uL. A260/A280 = 1.40. Dilution specified: 20 uL sample + 80 uL diluent.

Conclusions: From these measurements, it appears that for a single-stranded DNA oligomer sample with A260 value of about 1.7, corresponding to a DNA concentration of about 50 ng/uL, the technically achievable precision is about 0.1-0.2 ng/uL for repeat measurements on an undisturbed sample, and on the order of 0,5 ng/uL for repeat measurements when the sample is removed from and then replaced in the photometer. The largest variations were observed when turning the cuvette 180 degrees and measuring with the cuvette in opposite orientations, with the largest variation between measurments within that series of measurements (measurements #12-#15) being 1,3 ng/uL. For all the measurements performed, the range of measurements was 1,7 ng/uL. Thus, for measurements of DNA oligomer concentrations it seems prudent to report the measurements with an expected error of at least 2 ng/uL. These numbers assume a conversion factor of 1 A260 = 30 ug/mL. For consistency, all cuvettes used for a series, including the blanking cuvette, should be oriented in the same direction when performing measurements.

Note 1: The solution with expected concentration 10 uM was prepared previously by mixing 10 uL of a solution prepared from dry DNA received from Macrogen Inc. by resuspension in 220 uL DSBio NF water for an expected concentration of 100 pmol/uL. A concentration of 5 uM gives the following expected concentration by mass: 5 * 10^-6 mol/L * ~ 6000 g/mol = 0.03 g/L (0.03 ug/uL, 30 ug/mL, 30 ng/uL). Using a conversion factor of 1 A260 = 30 ug/mL, the expected A260 is thus ~ 1.0 A260. The photometric measuring range according to the Biophotometer manual is up to 2.6A at 260 nm when using Eppendorf UVette cuvettes.

Note 2: The solution with expected concentration 10 uM was prepared by resuspension of dry ITS4 DNA with the appropriate amomunt of NF water, in the same fashion as for the ITS1 solution.

=Centrifugation=

Typical centrifugal forces specified in protocols for spin column kits are 14 000 - 18 000 g. Examples:

NOTE: Conversion factor between rcf (g) and speed (rpm) depends on the centrifuge/rotor.

*QIAquick PCR purification kit: 17900 g (13000 rpm)
*Dongsheng Biotech PCR PCR and DNA Fragment Purification Kit: 12 000 rpm
*Promega Wizard Plus SV Miniprep purification system: ~14 000 g

==Eppendorf Minispin/Minispin Plus==

*Max centrifugal force: 12 100 g (Minispin) / 14 000 g (Minispin Plus)
*Max RPM: 13 400 (Minispin) / 14 500 (Minispin Plus)
*Weight without rotor: 3.7 kg
*Power consumption: 0.45 A at 230V (minispin)/ 0.60 A at 230 V (Minispin Plus)
*Dimensions: 22.5 (Width)/ 24.0 (Depth) / 12.0 (Height) cm

*Minispin 230 V model no.: 022620151
*Minispin 110 V model no.: 022620100

==Eppendorf 5415D==

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

*Max speed: 13 200 rpm
*Max centrifugal force: 16 110 g
*Max load: 24 x 2,0 mL or 36 x 0,5 mL tubes (rotor dependent)
*Power requirement: 180 W
*Weight without rotor: 8.5 kg
*Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm

=Weighing=

Background reading:

*Weighing scale terminology: http://www.awscales.com/support/terminology
*Good Weighing Practice brochure (Mettler Toledo): https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/MettlerToledo/11793161_GWPbrochure.pdf
*Good weighing practice in pharmaceutical industry (Radwag): http://radwag.com/pliki/artykuly/good_weighing_practice_in_pharmaceutical_industry.pdf
*Proper weighing with laboratory balances: http://www.masontechnology.ie/files/documents/ME108.pdf
*EMFR weighing principle: http://www.wipotec.com/en/company/weighing-principle/

==mg scales==

*Ohaus Scout (SPU123). (NOK 3 562,50 inkl. MVA : https://www.vektekspert.no/laboratorievekt-ohaus-scout.html

Kern EMB 100-3. 2761 NOK inkl MVA: https://www.vektekspert.no/presisjonsvekt-kern-emb.html

Gempro 300 (1748 NOK inkl MVA): https://www.vektekspert.no/praecisionsvaegt-gempro-300.html

Kern TAB 20-3 lommevekt (998,75 NOK inkl MVA). Non-automatic, not suitable for dynamic weighing: https://www.vektekspert.no/lommevaegt-kern-tab.html#ptabs

==Analytical balances==

Kern ADB 200-4 (NOK 11 961,25 inkl. MVA): https://www.vektekspert.no/analysevaegt-kern-adb.html

Ohaus Pioneer PA114CM (NOK 12 475,00 inkl. MVA): https://www.vektekspert.no/analysevekt-ohaus-pioneer.html

BioHackerLab/Equipment

2016-08-14T10:26:44Z

Jarlemag: /* Weighing */

BioHackerLab/Equipment

2016-08-14T10:11:45Z

Jarlemag:

BioHackerLab/Equipment

2016-08-13T11:13:36Z

Jarlemag:

BioHackerLab/Equipment

2016-08-13T10:37:07Z

Jarlemag:

BioHackerLab/Equipment

2016-08-13T10:36:37Z

Jarlemag:

Biolaboratoriet

2016-08-13T10:33:32Z

Jarlemag: /* Bøker */

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

=Planlagte meetups=

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x Mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

Budding Yeast: A Laboratory Manual: http://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=1071

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

BioHackerLab/Equipment

2016-08-13T10:28:23Z

Jarlemag:

BioHackerLab/Equipment

2016-08-13T10:19:21Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:54:41Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:54:28Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:54:19Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:53:54Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:43:37Z

Jarlemag:

BioHackerLab/Equipment

2016-08-12T20:41:41Z

Jarlemag:

Biolaboratoriet

2016-08-11T21:47:47Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

=Planlagte meetups=

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x Mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

http://www.instructables.com/id/DIY-BioPrinter/

http://opentrons.com/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

Biolaboratoriet

2016-08-11T21:45:39Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

=Planlagte meetups=

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x Mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

http://www.gaudi.ch/OpenDrop/

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

BioHackerLab/Protokoller

2016-08-10T20:53:03Z

Jarlemag:

=Resuspendering av DNA-primere=

Ta røret med tørket DNA ut av pakken. Kontroller at ID og DNA-sekvens oppgitt på røret stemmer med bestillingen.

Plasser røret i en mikrosentrifuge og sentrifuger i ca. 30 sekunder.

Tilsett NF-vann til konsentrasjon etter oppløsning lik 100 pmol/uL (0.1 mM, 100 uM). Se siden av røret eller medfølgende dokumentasjon fra leverandør for total mengde DNA i røret og/eller mengde vann som må tilsettes for endelig konsentrasjon ~100 pmol/uL

Bland ved å knipse på og riste flasken, eller med en vortex-mikser. Sentrifuger til slutt røret igjen.

Oppbevar fryst, fortrinnsvis ved -20C.

Før bruk til PCR vil det være hensiktsmessig å foreta ytterligere 10X fortynning til 10 pmol/uL (10 uM) i et eget rør, som også kan oppbevares fryst og brukes flere ganger. Dette reduserer også behovet for å åpne hovedrøret og reduserer risikoen for kontaminasjon av dette.

Se også https://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html?

Om mulig kontroller konsentrasjonen vha. spektrofotometri. Se http://www.promega.com/a/apps/biomath/index.html?calc=odConvert

=DNA extraction=

Blount, Driessen & Ellis: GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA. Scientific Reports 6, Article number: 26863 (2016). http://www.nature.com/articles/srep26863

Strawberry crude DNA extraction: http://www.stevespanglerscience.com/lab/experiments/strawberry-dna/

=DNA quantification=

Reference sample: https://crm.irmm.jrc.ec.europa.eu/p/ERM-AD442k

==Background==

The main methods for determiniing nucleic acid concentrations are UV photometry measuring absorbance at 260 nm (A260) and fluorometry. Note that different sources may describe different conversion factors for conversion from A260 to mass concentration. For oligomers, a more accurate DNA concentration value may be achieved by estimating the sequence-dependent extinction coefficient by calculation.

https://www.scripps.edu/california/research/dna-protein-research/forms/biopolymercalc2.html

http://biotools.nubic.northwestern.edu/OligoCalc.html

http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full

http://www.promega.com/a/apps/biomath/

https://no.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/

http://www.ogt.co.uk/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality

https://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/09%20UV%20Absorption.pdf

https://www.idtdna.com/pages/support/technical-vault/reading-room/quick-reference/quick-reference/2011/06/02/molar-extinction-coefficient

http://www.endmemo.com/bio/OD260.php

=Elektroforese=

Se også: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_SeaKem_LE_Agarose_-_Protocol.pdf

'''OBS: Les gjennom hele protokollen før gjennomførelse for å bli kjent med relevant sikkerhetsinformasjon.'''

==Utstyr==

'''OBS: Kontroller utstyret for skader og slitasje før bruk. Ikke fortsett dersom elektroforesekammer, ledninger eller strømforsyning viser tegn til skade.'''

*Elektroforesekammer. Eks. Carolina Deluxe Gel Chamber.
*Strømforsyning. Eks. BioRad PowerPac Basic
*Automatpipette, 1-10 uL.
*Vekt, 0.1 g eller høyere oppløsning.
*Mikrobølgeovn
*Varmebeskyttende hansker eller annen håndbeskyttelse
*Målesylinder, 50 mL eller større
*Glassflaske til agaroseløsning, 100 mL eller større (må passe i mikrobølgeovn)
*Glassflaske til TAE-bufferløsning, 250 mL eller større
*Transilluminator, eks. DarkReader blue light.
*Stekespade eller lignende for å løfte agarosegel

Avfallsbeholder for pipettespisser og tørt avfall
Avfallsbeholder for brukt buffer/flytende avfall

Forbruksvarer:
*Agarose for elektroforese, eks. SeaKem LE agarose. Ca. 0.5 g/gel.
*TAE 10x buffer. Ca. 30 mL.
*Destillert vann: Ca. 300 mL.
*DNA-fargestoff, eks. GelGreen. Ca 5 uL.
*Pipettespisser, 1-10 uL.
*Veieskip.
*Tørkepapir
*DNA ladder
*Loading dye

==Støping av agarose-gel==

Om nødvendig, lag TAE buffer i brukskonsentrasjon ved å fortynne konsentrert buffer i destillert vann. For å lage 1x TAE buffer fra 10x TAE buffer, bland 1 del TAE buffer og 9 deler destillert vann. TAE buffer er irriterende. Bruk hansker ved håndtering av konsentrert buffer.

Tilsett gradvis ~1 v/v % agarose til 1x TAE buffer (~1g til 100 mL, f.eks 0.5g for 50 mL) i en flaske med volum minst to ganger volumet av løsningen, fortrinnsvis under omrøring med magnetrører - eventuelt rør for hånd med glasstav eller ved å bevege flasken.

'''OBS: Flasken må ikke være lufttett!'''

Varm løsningen i mikrobølgeovn på høy styrke til løsningen koker (ca. ett minutt). La løsningen koke i ca. 30 sekunder. Reduser varmeeffekten eller skru av ovnen dersom løsningen koker over.

Fjern flasken fra mikrobølgeovnen. Bland løsningen ved å bevege flasken '''forsiktig''' i en roterende bevegelse for hånd, eller med en magnetrører. Utvis forsiktighet for å unngå sprut. Kontroller at agarose-løsningen er klar og uten synlige partikler. Dersom det er partikler og uløst agarose i løsningen, kok løsningen igjen.

'''OBS: Bruk øyebeskyttelse! Fare for støtkoking og sprut også etter at oppvarmingen har opphørt. Flasken er varm. Bruk varmebeskyttende hansker eller annen håndbeskyttelse ved håndtering av flaske. Bruk fortrinnsvis labfrakk eller langarmede klær og unngå eksponering av bar hud. Fare for forbrenning ved søl eller sprut.'''

Tilsett DNA-fargestoff i henhold til konsentrasjonsangivelse. F.eks, for 10 000x konsentrert fargestoff, tilsett 1 uL fargestoff per 10 mL agarose løsning (5 uL for 50 mL).

Plasser et gel-støpekar på tvers av lengderetningen i et elektroforesekar og plasser en brønnkam i støpekaret. Hell varm agaroseløsning i kammeret og la stå til gel'en er størknet (ca. 30-60 minutter).

'''OBS: Elektroforesekaret skal være frakoblet fra strømforsyning når gel'en støpes.'''

Ta ut brønnkammen og snu støpekaret slik at prøvebrønnene er nærmest den sorte (postive) elektroden.

Hell 1x TAE buffer i elektroforesekaret slik at gel'en er dekket av buffer.

==Elektroforese==

*Tilsett prøvene i prøvebrønnene.
*Sett på lokket på elektroforesekaret.
*Kontroller at området rundt elektroforesekaret og strømforsyningen er tørt. Tørk bort eventuell væske.
*Koble ledningene til strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.

'''OBS: Strømforsyningen skal være avslått når ledningene kobles til!'''

*Skru på strømforsyningen.
*Juster til ønsket spenning og skru på spenningen.
*La elektroforesen foregå uforstyrret. '''Ikke rør kammeret eller ledningene så lenge spenningen er på. Dersom det oppstår tegn til lekkasje fra kammeret, avslutt elektroforesen umiddelbart uten å komme i kontakt med væsken.'''
*Skru av spenningen på strømforsyningen.
*Skru av strømforsyningen.
*Koble ledningene fra strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.
*Ta av lokket på elektroforesekaret.
*FLytt agarosegeleen forsiktig til en transilluminator ved hjelp av en stekespade e.l.
*Avhend agarose-gel, bufferløsning og forbruksmateriell på forsvarlig vis etter bruk. Skyll elektroforesekammer og glassutstyr med destillert vann.

Se også: https://www.addgene.org/plasmid-protocols/gel-electrophoresis/

http://www.methodbook.net/dna/agarogel.html

Typisk prøvevolum kan være 6 uL, f.eks 5 uL PCR-produkt + 1 uL 6x loading dye, eller 4 uL vann, 1 uL DNA ladder og 1 uL 6x loading dye.

=PCR=

Typiske reaksjonsvolum er 20 uL og 50 uL. Gitt en konsentrasjon i primerløsningen lik 10 uM vil i de to tilfellene tilsetting av henholdsvis 1 uL og 2.5 uL av hver primer gi en endelig kosentrasjon av hver primer lik 0.5 uM i reaksjonsblandingen.

http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Typisk vil en ta ut ca. 5 uL av reaksjonsblandingen til elektroforese-analyse etter PCR.

Se også https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

==Detection of D1S80 Repeat Polymorphism by PCR==

See https://www.dnalc.org/files/pdf/forensicprofchip_d1s80_protocol.pdf

==Collection of genomic DNA by buccal swabs==

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566681/pdf/envhper00512-0045.pdf

==PTC tasting ability genotyping==

https://www.snpedia.com/index.php/Rs713598

https://www.ncbi.nlm.nih.gov/pubmed/12595690?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181941/

http://www.ncbi.nlm.nih.gov/gene?cmd=retrieve&dopt=default&rn=1&list_uids=5726

http://bioinformatics.dnalc.org/ptc/animation/pdf/ptc.pdf

==Sex determination by PCR==

See https://en.wikipedia.org/wiki/Amelogenin#Application_in_sex_determination and Fracnes et al.: Clin Chim Acta. 2007 Nov-Dec;386(1-2):53-6. Epub 2007 Jul 31.
Amelogenin test: From forensics to quality control in clinical and biochemical genomics.. (http://www.sciencedirect.com/science/article/pii/S0009898107003889)

Primers:

*AME-F: 5'-ctgatggttggcctcaagcctgtg-3'
*AME-R: 5'-taaagagattcattaacttgactg-3'

Expected results:

*If male: 1 band of 977 bp + 1 band of 790 bp
*If female: 1 band of 977 bp

==DNA fingerprinting==

===CODIS===

The Combined DNA Index System (CODIS) is a FBI program to support law enforcement by DNA-based identification. CODIS defines standards for DNA fingerprinting and a selection of genetic loci to be analyzed as part of DNA fingerprinting. The 13 CODIS core loci per 2016 are:

*CSF1PO
*FGA
*THO1
*TPOX
*VWA
*D3S1358
*D5S818
*D7S820
*D8S1179
*D13S317
*D16S539
*D18S51
*D21S11

In addition, the Amelogenin (AMEL) locus is used for sex determination.

For information on each locus, see http://www.cstl.nist.gov/strbase/

See also http://www.cstl.nist.gov/strbase/coreSTRs.htm

7 additional loci will be required from January 1 2017.

http://isogg.org/wiki/CODIS

https://en.wikipedia.org/wiki/Combined_DNA_Index_System

http://www.cstl.nist.gov/biotech/strbase/fbicore.htm

http://www.cybertory.org/resources/CODIS/

Estimated cost for CODIS primer set as described by cybertory.org, with synthesis by Macrogen Inc.: 68.2 EUR / ~75 USD / ~ 650 NOK + shipping.

===European set===

The European Standard Set (ESS) consists of the following STR loci: FGA, TH01, VWA, D1S1656, D2S441, D3S1358, D8S1179, D10S1248, D12S391, D18S51, D21S11, D22S1045

Additional Loci commonly found in European STR kits include: D2S1338, D16S539, D19S433, SE33, Amelogenin

==Colony PCR==

1. From an agar plate, select one or several colonies for colony PCR. For each colony selected, pick a small amount of colony material using a sterile pipette tip, and dissolve in 50 uL H2O. Reseed a new agar plate with 5 uL of the resulting solution(s) each in a separate spot, keeping the spots separate and noting the location of each. Alternatively, use 5 uL to inoculate a liquid culture.

Incubate the dissolved colony material at 96 C for 10 min to release DNA.

Use 1 uL of the heat-treated solution as template for PCR.

==ITS1 + ITS 4 yeast==

===Background===

See Esteve-Zarzoso et al.: ''Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers''. (http://www.ncbi.nlm.nih.gov/pubmed/10028278)

[https://www.researchgate.net/publication/13261852_Esteve-Zarzoso_B_Belloch_C_Uruburu_F_Querol_A_Identification_of_yeasts_by_RFLP_analysis_of_the_58S_rRNA_gene_and_the_two_ribosomal_internal_transcribed_spacers_Int_J_Syst_Bacteriol_49_329-337 Artikkel tilgjengelig via ResearchGate]

See also http://sites.biology.duke.edu/fungi/mycolab/primers.htm

[[Fil:ITS1 ITS4 insilico UCSC.png|miniatyr|høyre|Screenshot of in silico PCR result using the UCSC in silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr) with the primer sequences ITS1 (5' TCCGTAGGTGAACCTGCGG 3') and ITS4 (5' TCCTCCGCTTATTGATATGC 3'), selecting the S. cerevisae April 2011 (SacCer_Apr2011/sacCer3) genome assembly as the template. The result shows two expected PCR products/target regions with identical sequences. For each, the line starting with a > specifies the target location in the assembly, the target region size/expected product size, and the primer sequences, separated by spaces. The subsequent lines show the target region/expected product sequence, with the sequence of the forward primer (ITS1) and the sequence matching the reverse primer (ITS4) in capitals. The target region size/expected product size (841bp) includes the primers.]]

===PCR setup===

'''According to Esteve-Zarzoso et al.:'''

Reaction volume: 100 uL

Primer concentrations: 0.5 uM each

Forward (FWD) primer (ITS1): 5' TCCGTAGGTGAACCTGCGG 3'

Reverse (REV) primer (ITS4): 5' TCCTCCGCTTATTGATATGC 3'

(Start and end of expected product sequence = FWD primer + reverse complement of reverse primer: TCCGTAGGTGAACCTGCGG-GCATATCAATAAGCGGAGGA)

Template: Fresh yeast colony material.

Template preparation: 95 C for 15 min.

Initial denaturation: 95C for 5 min

Repeated cycles:
*Denaturation: 94 C for 1 min
*Anneal: 55.5 C for 2 min
*Extension 72C for 2 min

x 35 cycles

Final extension: 72C for 10 min

Total hold time: 190 min

Estimated program duration with OpenPCR: ~4 hr.

'''Alternative setup and reduced duration PCR program:'''

Reaction volume: 50 uL

Template: The protocol has been tested with dry yeast, see template preparation below. The protocol may also be attempted with fresh store-bought yeast, liquid yeast culture or yeast agar colony material as the template source.

PCR setup otherwise as original protocol.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 35 cycles

Step durations and temperatures are according to recommendations for Dongsheng Taq polymerase. Annealing temperature according to original protocol.

Final extension: 72 C for 10 min

Total hold time: 83 min

Run time with OpenPCR: ~ 2h 20 min.

'''Template preparation:'''

Dissolve 0.1g dry yeast (Idun tørrgjær) in 10 mL dH20. Mix well. Pipette 50 uL (-> ~0.0005g, 0.5mg yeast powder by dry weight) into a microsentrifuge tube or PCR tube and incubate at 98C for 10 minutes. Use 1-2 uL (-> ~0,00002g = 0,02mg = 20 ug yeast powder by dry weight for 2 uL) of the supernatant as template.

I tørrvekt kan det forventes at DNA utgjør i størrelsesorden 1% av cellemassen. ([http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/ ref])
20 ug tørrmasse vil da gi anslagsvis ~0.2 ug = 200 ng DNA.

DNA har molekylær vekt lik ca. 650 g/mol per basepar. ([https://www.neb.com/tools-and-resources/usage-guidelines/nucleic-acid-data ref]). S. cerevisae-genomet består av ca. 12,156*106 basepar. Basert på dette får vi da

200 *10-9 g DNA/ 12,156*106 bp) * (650 g/mol * bp) ~ 2,5*10-17 mol genomkopier.

Et mol er lik 6 * 1023. Vi har da

2,5 *10-17 mol * 6 * 1023 / mol ~ 1,5*107 (15 millioner) kopier.

S. Cerevisae har ca. 100-200 kopier av hvert rRNA-gen. Vi får da

15 millioner genomkopier * (~100 genkopier/genom) ~ 1,5 milliarder genkopier.

'''Extra reduced duration PCR program:'''

Tested 10.jul.2016. Amplification was successful for 1 of 2 samples.
Based on the results, further shortening to 25 cycles may be attempted.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 30 cycles

No final extension.

Total hold time: 63 min

Run time with OpenPCR: ~112 min. (1 h 52 minutes).

===Expected results===

'''Information from literature:'''

Expected PCR product size for ''S. cerevisiae'' (Strains CECT 1942 /ATCC 18824, CECT 1971) as reported by Esteve-Zarzoso et al: 880 bp.

'''Information from ''in silico'' PCR and published genomic sequence data:'''

[[Fil:ITS1 ITS4 S288C BLAST result.png|miniatyr|miniatyr|Graphic representation of BLAST result showing the location of the PCR target regions for the ITS1 and ITS4 primers in the S. cerevisae S288C genome (assembly R64, GenBank accession GCA_000146045.2). The query sequence is the expected PCR product sequence returned by the UCSC in silico PCR tool. While there are two target regions, there is only one expected product sequence as the sequence at and between the primer binding sites is identical for the two target regions. The locations of the target regions are shown as grey bands along the bottom, with the text "Query_185011" above each band. The left-pointing arrowing on each band indicates that the sequence of the query matches the sequence of the reverse strand as defined in the genome sequence file.]]

Expected fragment sizes as returned by in silico PCR at https://genome.ucsc.edu/cgi-bin/hgPcr (April 2011 assembly): 2 fragments of 841 bp each ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500810913_rmZAxNnexxkDaK66dHwLt86MeALA&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TCCGTAGGTGAACCTGCGG&wp_r=TCCTCCGCTTATTGATATGC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link]). The reported locations are chrXII:464259-465099 chrXII:455122-455962

The sequences of the two expected products as identified by in silico PCR above are identical. BLAST of the sequence against the R64 Assembly ([http://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/ GenBank GCA_000146045.2]) returns two matches, both on chromosome XII: Range 1: 455122 to 455962; and Range 2: 464259 to 465099. Both with 841/841 (100%) identities and zero gaps.

The genome assembly used for the in silico PCR described above is described as "Apr. 2011 (SacCer_Apr2011/sacCer3)". Although the relation or differences between this assembly/description and the R64 assembly have not been determined, the result of the BLAST search shows that the nucleotide positions on chromsome XII as reported in the ''in silico'' PCR result correspond to those of the R64 genome assembly and that there are no differences in the sequences of these regions. ([http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&QUERY=TCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTG%0AGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAG%0ATGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTG%0ATTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTT%0AGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTC%0AAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCT%0ACGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATC%0ATTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAAC%0AATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTT%0ATTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAAC%0ATGCGGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACA%0AATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATATCAATAAGCGGAGG%0AA&JOB_TITLE=Nucleotide%20Sequence%20%28841%20letters%29&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=Assembly&DATABASE=genomic/559292/GCF_000146045.2&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=4932&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=R&FILTER=m&WWW_BLAST_TYPE=newblast&EQ_MENU=Enter%20organism%20name%20or%20id--completions%20will%20be%20suggested&SHOW_OVERVIEW=true&SHOW_LINKOUT=true&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=true&NEW_VIEW=false&NCBI_GI=false&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&SHOW_CDS_FEATURE=false&OLD_BLAST=false direct link to search query])

(The sequence for S288C chromosome XII alone is accesible at: http://www.ncbi.nlm.nih.gov/nuccore/NC_001144.5)

[http://www.yeastgenome.org/browse/?loc=chrXII%3A463459..466018&tracks=DNA%2CAll%20Annotated%20Sequence%20Features&highlight=chrXII%3A464259..465099 Size & location of expected fragment 1, shown as highlighted area in SGD genome browser.]

[http://www.yeastgenome.org/browse/?loc=chrXII%3A454515..457224&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight=chrXII%3A455122..455962 Size & location of expected fragment 2, shown as highlighted area in SGD genome browser]

[[Fil:RDN582-region.png|700px|sentrer|Representation of the yeast genome region centered on the RDN58-2 rRNA gene, as shown in the SGD genome browser using the latest version of the S. cerevisae S288C genome (assembly R64). The S. cerevisae genome contains 100-200 repeats of the genes shown. The yellow region represents the target area for the ITS1-ITS4 primer combination in this location.]]

The target region spanning nucleotide positions 455122 to 455962 covers the 5.8S ribosomal RNA (rRNA) gene RDN58-1 (455414..455571) and the non-coding regions ITS2-1 and ITS1-1 which flank RDN58-1. Likewise, the target region spanning nucleotide positions 464259 to 465099 covers the 5.8S rRNA gene RDN58-2 (464551..464708) and the flanking non-coding regions ITS2-2 and ITS1-2.

SGD entry for RDN58-1: http://www.yeastgenome.org/locus/S000006488/overview

SGD entry for RDN58-2: http://www.yeastgenome.org/locus/S000006489/overview

RDN58-1 and RDN58-2 are contained within the [http://www.yeastgenome.org/locus/S000029411/overview RDN1 locus]. Although the sequence included in assembly R64 only indicates two target regions for the ITS1-ITS4 primer combination, the RDN1 locus in actuality represents a 1-2Mbp repeating region containing 100-200 rDNA repeats ([http://www.yeastgenome.org/locus/S000029411/overview ref]). Thus the actual number of targets can be expected to be in this range, and some sequence variation among these is likely ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665781/ ref]).

'''Expected PCR product sequence (841 bp):''' (NB: Whitespace is included below for easier viewing, adding extra characters)

Esteve-Zarzoso et al. used the restriction enzymes CfoI, HaeIII, and HinfI to cleave PCR product DNA and perform Restriction Fragment Length Polymorphism (RFLP) analysis. The sequence below contains 3 binding sites for CfoI (GCGC) CfoI, 3 binding sites for HaeIII (GGCC) and 3 binding sites for HinfI (GANTC, where N is any nucleotide). The sequence also contains an EcoRI binding site (GAATTC, bolded).

Esteve-Zarzoso reports the restriction fragment sizes for digestion with CfoI, HaeIII and HinfI as 385 + 365 (= 750), 320 + 230 + 180 + 150 (= 880) and 365 + 155 ( = 520), respectively (fragments smaller than 50 bp not included or reported).

Based on the sequence below, the expected fragment sizes from digestion are (http://www.restrictionmapper.org/):

*EcoRI 469 + 372 (= 841)
*CfoI: 363 + 334 + 134 + 10 (= 841)
*HaeIII: 311 + 230 + 172 + 128 (= 841)
*HinfI: 362 + 355 + 116 + 8 ( = 841)

<code>TCCGTAGGTGAACCTGCGGaaggatcattaaagaaatttaataattttga
aaatggatttttttgttttggcaagagcatgagagcttttactgggcaag
aagacaagagatggagagtccagccgggcctgcgcttaagtgcgcggtct
tgctaggcttgtaagtttctttcttgctattccaaacggtgagagatttc
tgtgcttttgttataggacaattaaaaccgtttcaatacaacacactgtg
gagttttcatatctttgcaactttttctttgggcattcgagcaatcgggg
cccagaggtaacaaacacaaacaattttatctattcattaaatttttgtc
aaaaacaagaattttcgtaactggaaattttaaaatattaaaaactttca
acaacggatctcttggttctcgcatcgatgaagaacgcagcgaaatgcga
tacgtaatgtgaattgca'''gaattc'''cgtgaatcatcgaatctttgaacgca
cattgcgccccttggtattccagggggcatgcctgtttgagcgtcatttc
cttctcaaacattctgtttggtagtgagtgatactctttggagttaactt
gaaattgctggccttttcattggatgttttttttccaaagagaggtttct
ctgcgtgcttgaggtataatgcaagtacggtcgttttaggttttaccaac
tgcggctaatctttttttatactgagcgtattggaacgttatcgataaga
agagagcgtctaggcgaacaatgttcttaaagtttgacctcaaatcaggt
aggagtacccgctgaacttaaGCATATCAATAAGCGGAGGA</code>

==V9D - LS266 yeast==

===Background===
"The primers V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used [to] amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA gene regions. These primers bind to conserved regions, with corresponding positions to Saccharomyces cerevisiae 18S (1609-1627) and 28S (287-266) rRNA genes." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

===PCR setup===

As described by Pryce (2010):

*Reaction volume: 50 uL
*Initial denaturation: 95 C for 9 min
*Repeated cycles:
*Denaturation: 95 c for 30s
*Anneal: 62C for 60s
*Extension: 72 C for 2 min
*Final extension: 72C for 5 min

x 33 (PCRS-B variant) or 35 (PCRS-D variant) cycles.

Total hold time: 136,5 min

===Expected results===
Expected fragment size: 1228 bp. ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500811567_RQun5ZzNd9dZSuQJcMHIctDo3PTE&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TTAAGTCCCTGCCCTTTGTA&wp_r=GCATTCCCAAACAACTCGACTC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link])

Expected fragment sizes after digestion by EcoRI: 628 + 600.

Expected PCR product sequence (S. cerevisiae):

<code>TTAcGTCCCTGCCCTTTGTAcacaccgcccgtcgctagtaccgattgaat
ggcttagtgaggcctcaggatctgcttagagaagggggcaactccatctc
agagcggagaatttggacaaacttggtcatttagaggaactaaaagtcgt
aacaaggtttccgtaggtgaacctgcggaaggatcattaaagaaatttaa
taattttgaaaatggatttttttgttttggcaagagcatgagagctttta
ctgggcaagaagacaagagatggagagtccagccgggcctgcgcttaagt
gcgcggtcttgctaggcttgtaagtttctttcttgctattccaaacggtg
agagatttctgtgcttttgttataggacaattaaaaccgtttcaatacaa
cacactgtggagttttcatatctttgcaactttttctttgggcattcgag
caatcggggcccagaggtaacaaacacaaacaattttatctattcattaa
atttttgtcaaaaacaagaattttcgtaactggaaattttaaaatattaa
aaactttcaacaacggatctcttggttctcgcatcgatgaagaacgcagc
gaaatgcgatacgtaatgtgaattgcagaattccgtgaatcatcgaatct
ttgaacgcacattgcgccccttggtattccagggggcatgcctgtttgag
cgtcatttccttctcaaacattctgtttggtagtgagtgatactctttgg
agttaacttgaaattgctggccttttcattggatgttttttttccaaaga
gaggtttctctgcgtgcttgaggtataatgcaagtacggtcgttttaggt
tttaccaactgcggctaatctttttttatactgagcgtattggaacgtta
tcgataagaagagagcgtctaggcgaacaatgttcttaaagtttgacctc
aaatcaggtaggagtacccgctgaacttaagcatatcaataagcggagga
aaagaaaccaaccgggattgccttagtaacggcgagtgaagcggcaaaag
ctcaaatttgaaatctggtaccttcggtgcccgagttgtaatttggagag
ggcaactttggggccgttccttgtctatgttccttggaacaggacgtcat
agagggtgagaatcccgtgtggcgaggagtgcggttctttgtaaagtgcc
ttcgaaGAGTCGAGTTGTTTGGGAATGC</code>

==MEATF + MEATR, animal meats==

===Background===

Lago et al. describe amplification and sequencing of the cytochrome b (cyt b) gene of several species using a set of degenerate primers. (European Food Research and Technology
March 2011, Volume 232, Issue 3, pp 509-515. http://link.springer.com/article/10.1007/s00217-010-1417-1)

===PCR setup===
Primers:
*MEAT F: CGAGGCCTMTAYTAYGG
*MEAT R: ATTGAKCGTAGGATTGCGTA

M denotes A or C. Y denotes C or T.

PCR program:

*Initial denaturation: 95C for 3 min
*Denaturation: 95 C for 30s
*Annealing: 50C for 30s
*Extension: 72 C for 30s

x 35 cycles.

*Final extension: 72C for 3 min

===Expected results===

Lago et al. report that DNA amplification with the primers MEAT F/R gave an amplicon of 555 bp in all tested species.

==PORCINE FWD + PORCINE REV, pork==

===Background===

See Ilhak and Arslan. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique. Turk. J. Vet. Anim. Sci. 2007; 31(3): 159-163: http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf

The porcine primers used by Ilhak and Arslan were designed based on Lahiff (2001). Mol Cell Probes. 2001 Feb;15(1):27-35. Species-specific PCR for the identification of ovine, porcine and chicken species in meta and bone meal (MBM).

See also GenBank accesion AF039170.1: http://www.ncbi.nlm.nih.gov/nuccore/AF039170

===PCR setup===

Primers:
*Porcine FWD: GCC TAA ATC TCC CCT CAA TGG TA
*Porcine REV: ATGAAAGAGGCAAATAGATTTTCG

*Reaction volume: 50 uL
*20 pmol of each primer

PCR program:
*Denaturation: 94 C 45 s
*Annealing: 58C 45 s
*Extension: 72C 90 s.

x 30 cycles.

===Electrophoresis===

15 uL PCR product, 1.5 % agarose at 100 V for 2 h.

===Expected results===

Ilhak and Arslan report a PCR product size of 212 bp for porcine meat.

=Lagring av bakterie-stammer i glycerol=

*1 Prepare a solution of 60 % v/v glycerol in water. (For 25 mL, mix 10 mL water and 15 mL glycerol)
*2 Add 400 uL 60 % glycerol solution and 800 uL of the culture to be stored in a cryogenic tube.
*3 Mix thoroughly!
*4 Place in 5 C refrigerator for 30 min, then move to -80 C freezer.

=Sekvenseringstjenester=

==GATC (lightrun)==
Primer specifications:
*Tm 52-58 C
*17-19 bp
*G or C at 3' end (max 3 Gs or Cs)
*maximum 4 identical sequential bp.

Preparation instructions:
*Add 5 uL template DNA (80-100 ng/uL plasmid DNA or 20-80 ng/uL purified PCR product) and 5 uL primer, 5 uM (5 pmol/uL) in one 1.5 mL tube.
*Drop off at GATC collection point or ship to: GATC Biotech AG. European Custom Sequencing Centre. Gotrfied-Hagen-Strasse 20. 51105 Köln.

==Macrogen==

Primer specifications:
*18-25 bp
*40-60 % GC
*Tm 55-60

Preparation instructions:
*Add 20 uL DNA (100 ng/uL plasmid or 50 ng/uL purified PCR product) to one tube. Add 20µl primer (10 pmol/uL) to a separate tube (?)
*Ship to: Macrogen Europe, IWO, Kamer IA3-195, Meibergdreef 39,1105 AZ Amsterdam Zuid-oost. Netherlands. Attention: J.S .Park

Preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

=Brukermanualer=

BioRad PowerPac Basic Power Supply: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006213.pdf

Biolaboratoriet

2016-08-10T20:51:07Z

Jarlemag:

Noen navneforslag:

BitLab? BioRaf? Biologiverksted? Biospace?

See also our chat channel #biohackers on bitraf.slack.com. To join, send yourself an invitation at https://bitraf.no/slack-invite/

= Planlegging =
Det har i det siste vært diskusjoner om å starte en egen lab for biologi på Bitraf. Bitraf har allerede medlemmer som jobber med hydroponics/aquaphonics og flere er interessert i ølbrygging. 18 Mai 2016 var [http://www.meetup.com/bitraf/events/230806525/ det første møtet] for folk som er interessert i å starte opp dette. Ønsket er å utvide foreningen med et lokale som egner seg for [https://en.wikipedia.org/wiki/Do-it-yourself_biology Gjør-det-selv-biologi] og Biologi-hacking som retter seg etter norsk lov og de etiske retningslinjene fremsatt av [https://diybio.org/codes/draft-diybio-code-of-ethics-from-european-congress/ European DIYbio Congress].

Summary from Bitraf's 1'st MeetUp:
https://drive.google.com/file/d/0B5j_-m_-t56rMXNHVVkwVEpUVjg/view

Courses and Equipment:
https://docs.google.com/spreadsheets/d/1M2TKaHVBQ3iLuV55A8JCwTCzPXx_aNDFoxyzZBgSM8o/edit#gid=0

=Avholdte meetups=

* 18. mai 2016: http://www.meetup.com/bitraf/events/230806525/

* 30.mai 2016: http://www.meetup.com/BioHcking/events/231697495/

* 13. juni 2016: http://www.meetup.com/bitraf/events/231692626/ - > Vi fikk besøk av Marius Øgaard fra Oslo Lifetech

* 27. juni 2016: Bruk av DNA-elektroforeseutstyret ble demonstrert, og de fleste fikk prøve pipettering av DNA til gel'en. Heikki presenterte utkast til brev til mulige sponsorer og tok i mot tilbakemeldinger.

[[Fil:Dsc 0069.jpg|miniatyr|sentrer|Resultat av gel-elektroforese demonstrert på meetup 27.06.16. Spor #1 og 4-7 fra venstre: Dongsheng Biotech 1kb ladder (~5 uL). Spor 2,3 og 8: Dongsheng Biotech 50bp ladder (~5 uL). Bildet er tatt av Heikki Sørum.]]

*11. juli 2016: http://www.meetup.com/bitraf/events/232455024/. We performed PCR using 3 different yeast samples. As it became pretty late, the PCR samples were put in the freezer for later electrophoresis and checking of the results. To have something to look at, we performed electrophoresis of samples from the previous two PCR runs. Update: The PCR samples were checked by electrophoresis July 24. 1 of 6 reactions was succesful. See https://bitraf.no/wiki/BioHackerLab/Experimental#11_Jul_2016_-_Bitraf_PCR_.233

=Planlagte meetups=

*Monday <strike>August 1</strike> August 8 2016: DIY Biolab: Using basic bioinformatics Tools; PyMOL: http://www.meetup.com/bitraf/events/232677504/

*Monday August 22: DIY Biolab: A visit from the UiO iGEM team + strawberry DNA extraction: https://www.meetup.com/bitraf/events/233059010/

= Videre jobbing =
De av deltakerne som ønsket å jobbe med dette videre delte seg i to grupper som skal jobbe med Mikrobiologi og Utstyr til labben. Mikrobiologi-gruppen har planer for å jobbe med ølbrygging og klassifisering av gjær med [http://openpcr.org/ Open PCR].

[[Fil:YeastPCR ITS ITS4 050716.jpg|miniatyr|sentrer|Result from PCR experiment 05 july 2016 to copy the 5.8S rRNA gene RDN58 and flanking ITS regions from yeast (S. cerevisae). Primers used were ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). Primers were supplied by Macrogen Inc. Primer target concentration: 0.5 uM each. From left: DSBio 1kb ladder (5 uL), DSBio 50bp ladder (5 uL), PCR sample 1 (10 uL), PCR sample 2 (10 uL), PCR negative control (no template) sample (~5-10 uL). Electrophoresis at 75V for ~45 min on 1 % agarose with GelGreen DNA stain. Visualized with DarkReader DR22 transilluminator. PCR performed 05.07.16 with OpenPCR and DongSheng Biotech Taq mix. Reaction volume 50 uL. Template source is store bought dry yeast (Idun tørrgjær). Template source was prepared by dissolving 0.1 g dry yeast in 10 mL distilled water, and incubating 50 uL of the resulting yeast solution in a PCR tube at 98C for 10 min in openPCR. For PCR sample 1 and 2, 1 uL and 2 uL of the supernatant after incubation was added to the reaction mix, respectively. PCR program was as follows: Initital denaturation: 94C for 3 min. Repeated cycles: Denaturation: 94C for 30s. Anneal: 55.5C for 30s. Extension: 72C for 1 min. Final extension: 72C for 10 min. 35 cycles. Total run time: ~2h 20 min. ]]

==Utstyr vi har==
* 1x OpenPCR
* Automatpipetter for områdene 1-10, 10-100 og 100-1000 uL. (2 sett)
*1x Mikrosentrifuge for 1.5 mL-rør
*1x Vortex mixer
*Elektroforese-kammer: 2x Carolina deluxe electrophoresis chamber
*Elektroforesestrømforsyning: 1x BioRad PowerPac Basic.
*1x DarkReader DR22A blue light transilluminator
*Mikrobølgeovn
*Mikroskop: 1x Bresser Erudite DLX (ikke på Bitraf for øyeblikket)
*Vanndestillasjonsapparat
*Vannbad
*Varmeplate med magnetrører
*Eppendorf Biophotometer 6131

[[Fil:OpenPCR.jpg|miniatyr|OpenPCR]]

[[Fil:Electrophoresis.jpg|miniatyr|Electrophoresis equipment: Carolina deluxe gel chamber and BioRad PowerPac power supply. Bottles with 10x TAE buffer and electrophoresis-grade agarose (small bottle) are also visible.]]

[[Fil:Pipettes.jpg|miniatyr|Set of micropipettes; 1-10, 10-100 and 100-1000 uL. LHP brand (Liquid Handling Products).]]

[[Fil:I016 Biophotometer.jpg|miniatyr|Eppendorf Biophotometer 6131. Used for quantification of nucleic acids.]]

== Utstyr vi ønsker oss ==
*pH-meter
* UV-kamera. (Hva brukes dette til? --[[Bruker:Jarlemag|Jarlemag]] ([[Brukerdiskusjon:Jarlemag|diskusjon]]) 22. jul. 2016 kl. 18:41 (UTC))
* Sentrifuge: Høyhastighetssentrifuge for 10 mL- og 50 mL-rør. Ex. Sorvall Biofuge Primo / Primo R.
* Fryser (ideelt -20 C)
* Kjøleskap
* Varmeskap med shaker. Eks: https://webshop.no.alere.com/222ds-benchtop-shaking-incubator-230v_1.aspx
*Varmeblokk
* Autoklav
* Lab-glass (flasker, rør m.m.)
*Målepipetter i glass
*Flere automatpipetter?
* Filter-utstyr
* Vekst-medier
* Qubit Fluorometer
* Stereo mikroskop med minimum 400X. Ideelt et fasekontrast (phase contrast) mikroskop så vi kan se bakterier uten å måtte bruke fargestoffer.
*Presisjonsvekt (F.eks Kern EMB 100-3: https://www.vektekspert.no/presisjonsvekt-kern-emb.html)
*Mikroplateleser, ex. Tecan Infinite 200

I tillegg trenger vi laboratoriemøbler/inventar som f.eks.:
* Stålbenk/utslagsvask
* Fumehood / labbenk
* Filter til Fumehood
*Oppvaskmaskin

==PCR prosjekt==

===Hva er PCR?===

*Polymerase chain reaction/polymerase kjedereaksjon: https://en.wikipedia.org/wiki/Polymerase_chain_reaction
*PCR brukes for å kopiere DNA. En PCR-maskin med reagenser er en "kopi-maskin" for DNA.
*PCR kan brukes til å kopiere opp (amplifisere) DNA fra naturlige kilder/biologisk materiale for videre bearbeidelse eller analyse.
*DNA-fragmenter med en kjent sekvens (rekkefølge på nukleotider, "bokstavene" i DNA) i hver ende velges ut og kopieres selektivt.
*Sammensetning og lengde av DNA-tråden mellom start- og sluttpunktet kan variere. Informajson om lengden av DNA-fragmentene (visualiseres ved gel-elektroforese), og om kopiering fant sted (positiv/negativ reaksjon) kan brukes til å gjøre enkle genetiske analyser.
*Opparbeidet DNA kan (gitt god nok mengde og kvalitet) sendes til nærmere analyse av DNA-sekvensen (sekvensering).

PCR visualization: https://www.youtube.com/watch?v=2KoLnIwoZKU

===Hvorfor gjøre PCR?===

*En aktivitet med relativt lav terskel, med begrenset behov for opplæring, gode muligheter for å lykkes, overkommelige kostnader og få risikomomenter.
*En klassisk molekylærbiologi-teknikk. I daglig bruk verden over. Uunnværlig for molekylærbiologisk forskning og medisinsk diagnostikk. Nobelpris-vinnende.
*Potensiale for praktiske anvendelser med allmenn interesse. F.eks kontroll av artsopprinnelse for matvarer.
===Hva trengs for PCR?===

'''Fast utstyr:'''
*PCR-maskin/thermocycler. Automatiserer temperaturegulering gjennom reaksjonsforløpet. Temperatur-regulering kan i teorien gjøres manuelt med vannbad ved ulike temperaturer, men dette blir temmelig langsomt og kjedelig
*Mikropipette(r). Fortrinnsvis minst én automatpipette med justerbart volum i området 1-10 μL.
*En eller flere flasker til agarose, ca. 250 mL. Glass eller varmebestandig plast, bør passe i mikrobølgeovn.
*Mikrobølgeovn til oppvarming av agarose, evt. annen varmekilde + magnetrører
*Gel-elektroforesekammer
*Strømforsyning
*Transilluminator m/filter og/eller filterbriller
*Bør ha: Mikrosentrifuge.
*Bør ha: Kjøleskap/fryser til oppbevaring av reagenser (PCR mastermix bør oppbevares frosset) og lage is
*Kjekt å ha: Vanndestillator
*Kjekt å ha: Liten isoporboks eller lignende til å ha is i, for kjøling av prøver under forberedelse.

'''Forbruksmaterialer:'''
*Eppendorf-rør (plastrør tilpasset mikrosentrifuge, ca. 1.5 mL)
*PCR-rør (plastrør tilpasset PCR-maskin, ca 0.5 mL)
*Pipette-spisser til automatpipette(r)

'''Reagenser/kjemikalier:'''
*PCR mastermix
*TAE/TBE buffer, konsentrert
*DNA-fargestoff (Riktig type med hensyn til transilluminator)
*DNA-ladder (Blanding av DNA-fragmenter med kjent lengde. Brukes som referanse for lengde/størrelse av DNA-fragmenter ved elektroforese.)
*DNA loading dye (Viskøs fargeblanding til utblanding av PCR-produkt før overføring til agarosegel. Kan "hjemmesnekres"?)
*Elektroforese-agarose
*DNA-primere (eksperiment-spesifikke)
*Vann, fortrinnsvis destillert. Evt. flaskevann med lavt mineralinnhold.

'''Sikkerhetsutstyr:'''
*Vernebriller
*Engangshansker
*Varmeisolerende hansker e.l. til håndtering av varm agarose
*Fortrinnsvis labfrakk

===Sikkerhetsmomenter:===
*Strøm gjennom elektroforesekammer (ca. 50-100 V)
*Varm agaroseløsning
*Støtkoking eller glasseksplosjon ved oppvarming av agarose i mikrobølgeovn. Unngås ved å begrense effekt/oppvarmingshastighet, begrense tid under oppvarming og aldri varme opp lukkede flasker/beholdere.
*Potensielt skadelige kjemikalier (eks. Ethidum-bromid, "ETBR") og fargestoff som krever bruk av transilluminator med UV-stråling bør unngås. Alternativer med lavere risiko og bedre miljøprofil bør brukes, f.eks "GelGreen" fargestoff (brukes med transilluminator med synlig blått lys, redusert fare for øye/hudskader).

===Avfallshåndtering:===
Avhenger av reagensvalg! Individuell vurdering må gjøres for hvert stoff. Generelt:
*Størknet agarose kastes som restavfall. Flytende agarose må ikke tømmes i avløp, da dette vil størkne ved avkjøling.
*Brukte bufferløsninger kan helles i avløp
*Forbruksmateriell av plast med eventuelle reagensrester kastes i restavfall
*Utstyr rengjøres med vann etter bruk

===Reagenser, mulige leverandører og prisoverslag:===

'''PCR-reagenser:'''

DongSheng Biotech:
http://dongshengbio.com/en/cpjs.asp?classname=PCR%20Products

Taq Mix: http://dongshengbio.com/en/xxcp.asp?id=330/ http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

Eksempel-bestilling (2014):

Product Name Cat No Description Qty Unit Unit Price Value

Taq Mix (2x) P2011 1 1ml $8.80 $8.80

Water, nuclease-free P9021 1 5x1ml $2.00 $2.00

PCR and DNA Fragment Purification Kit N1091 1 50preps $20.00 $20.00

50bp ladder M1041 1 50ug $16.00 $16.00

6xDNA Loading Dye M9041 1 5x1ml $4.60 $4.60

shipment viaFedEx $55.00

Total $106.40

'''DNA-fargestoff:'''

GelGreen:

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

GelGreen, Carolina.com: http://www.carolina.com/biotechnology-electrophoresis-reagents/gel-green/217305.pr?question= (ca. $64.50 + shipping / 150 uL 10 000 x konsentrasjon. Nok til ca. 30-40 agarose-geleer.)

Suggested protocols for working with GelGreen: http://embitec.com/downloads/Suggested_Protocols-GelGreen.pdf

GelRed-GelGreen Safety report: http://biotium.com/wp-content/uploads/2013/07/GR-GG-Safety.pdf

'''Elektroforese-buffer:'''

TAE (Tris/Acetate/EDTA)-buffer, Promega: https://no.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tae-buffer_-molecular-biology-grade-_tris_acetate_edta_/ (390 kr/1000 mL 10x konsentrasjon = 39 kr/L ferdig buffer)

TAE elektroforesebuffer 50 x, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/tae-elektroforesebuffer-50x (1 250 kr/ 500 mL 50x konsentrasjon = 50 kr/L ferdig buffer

TBE (Tris/Borate/EDTA)-buffer kan også brukes.

'''Elektroforese-agarose:'''

Agarose, 10 g, Frederiksen Scientific: http://no.frederiksen.eu/shop/product/agarose--10-g (kr 319 + frakt. Nok til ca. 10 agarose-gel'er (Gitt 50 mL 2 % agarose). Temmelig dyrt...Har tidligere kjøpt 100g på eBay for USD 40 + frakt.

'''DNA-primere:'''
Macrogen Inc:

Ca. 0.2 EUR/basepar (bp) x ca. 30 bp x 2 primere = ca. 12 EUR
Shipping ca 20
Sum ca. 30-40 EUR. (2013-priser)

===Artikler/protokoller og lesestoff===

*The PCR controls you must use: http://bitesizebio.com/4074/the-pcr-controls-you-must-use/
*Yaest colony PCR. Utvalg av protokoller @ OpenWetware: http://openwetware.org/wiki/Yeast_Colony_PCR
*http://www.jove.com/video/3998/polymerase-chain-reaction-basic-protocol-plus-troubleshooting
*http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*Simple And Reliable Procedure For PCR Amplification Of Genomic Dna From Yeast Cells Using Short Sequencing Primers: http://onlinelibrary.wiley.com/doi/10.1080/15216549700202551/pdf
*http://openwetware.org/wiki/Agarose_gel_electrophoresis
*http://no.frederiksen.eu/inspirasjon/biologi/bioteknologi/tips-og-raad
*http://www.naturfag.no/utstyrsbeskrivelse/vis.html?tid=709639
*https://seqcore.brcf.med.umich.edu/sites/default/files/html/pcr.html

DNA Learning Center Biology Animation Library - Polymerase Chain Reaction: https://www.dnalc.org/resources/animations/pcr.html

===Possible sub-projects/experiments===
In rough order of increasing difficulty/complexity?
*Electrophoresis demonstration/equipment test: Demonstrate/test equipment and reagents for agarose gel electrophoresis. Separate and visualize DNA fragments of known size (DNA ladder).
*PCR demonstration/equipment test: Demonstrate/test equipment and reagents for PCR and agarose gel electrophoresis. Amplify DNA fragment of known expected size from purified DNA or from biological material (yeast?). Visualize and determine size/length of the DNA fragment(s) by agarose gel electrophoresis.
*Animal tissue/foodstuff species identification: Demonstrate/test identification of DNA from a suspected/known species in raw or processed food (ex, horsemeat).

===DIY PCR-maskiner - Eksisterende design og prosjekter===

http://openpcr.org/

https://www.kickstarter.com/projects/563115656/3d-printer-into-pcr-machine-conversion

Arduino PCR thermal cycler for under $85: http://www.instructables.com/id/Arduino-PCR-thermal-cycler-for-under-85/?ALLSTEPS

"Coffee Cup PCR": http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/?ALLSTEPS

http://www.popsci.com/diy/article/2013-04/gene-machine

===Tilgjengelige primere===

{| class="wikitable"
|-
! Navn !! Sekvens !! Beskrivelse !! Lengde !! Templat
|-
| Ec_lld_Rev || GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc || REV-primer for E. coli lld promoter + RBS. Se http://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results || 52 || E. coli
|-
| Ec_lld_FWD || GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt || FWD-primer for E. coli lld promoter + RBS. || 55 || E. coli
|-
| Fd2trim || GAGTTTGATCATGGCTCAG || || || Wide-range bacterial.
|-
| Porcine FWD + Porcine REV || || [2]
|}

MEATF + MEATR [2]

HorseSSR-FWD + HorseSSR-REV [2]:

FWD: 5'-TTC TGC TCT GGG TGT GCT ACT T-3' (22mer)
REV: 5'-CTA CTT CAG CCA GAT CAG GC-3' (20mer)

S-D-Bact-0515-a-A-19 + S-D-Bact-0341-b-S-17 [1]

S-D-Bact-0515-a-A-19: 5'-TTA CCG CGG CTG CTG GCA C-3' (19mer)
S-D-Bact-0341-b-S-17: 5'-CCT ACG GGN GGC WGC AG-3' (17mer)

[1]: See http://openwetware.org/wiki/User:Jarle_Pahr/16S_RNA

[2]: See http://openwetware.org/wiki/User:Jarle_Pahr/Meat

===Primers of interest / Shopping list ===

'''Yeasts:'''

"V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used to amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA regions." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

ITS 1 (5' TCCGTAGGTGAACCTGCGG 3') + ITS4 (5' TCCTCCGCTTATTGATATGC 3'). " In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts." (http://www.ncbi.nlm.nih.gov/pubmed/10028278, full text available through ResearchGate).

ITS1 and ITS4 available (10 USD for 100 reactions) from The ODIN: http://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

'''E. coli:'''

rrnB p1_74bp_FWD_R caaccggtgttgcgcggtcagaaaatta
rrnB p1_74bp_REV_R gtacatgtagtggtggcgcattatagg

Gives a short fragment.

'''pSB-M1g/pJP-1 plasmids:'''

pSB-SeqA/GFP-END-LVA-REV. Sequencing of and/or PCR demonstration using plasmid pSB-M1g. PCR from pSB-M1g without adding LVA tag should give fragment of aprox. 800 bp (size of GFP ORF + ~ 1 bp upstream).

pSB-SeqA: tgcaagaagcggatacag

GFP-END-LVA-REV: agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgctttgtatagttcatccatgcc (LVA sequence can be removed from the 5' end).

For PCR from pJP-1, replace pSB-seqA with pJP-1_seq5 (binding site is upstream of AgeI, as such this site is preserved from pSB-mg1, and this primer combination can also be used with pSB-mg1 for a longer PCR fragment)

=HMS=

http://www.ncbi.nlm.nih.gov/books/NBK55884/

==El-sikkerhet==
https://www.physics.ohio-state.edu/~p616/safety/fatal_current.html

==Ved nødsfall==

Giftinformasjonsentralen, døgnåpen vakttelefon: 22 59 13 00.

Legevakt: 116 117

==Avfallshåndtering==

'''Hva er farlig avfall?''' Se [https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12 Forskrift om gjenvinning og behandling av avfall (avfallsforskriften) kapittel 11, Farlig avfall.]I henhold til forskriftens §11-2 er farlig avfall definert som

* a) avfall som skal klassifiseres som farlig i henhold til vedlegg 1 til kapitlet,

* b) annet avfall som skal klassifiseres som farlig i henhold til vedlegg 2 nr. 1 til kapitlet.

'''Avfallsbesitter har ansvaret for å vurdere om avfallet omfattes av bestemmelsene i dette kapitlet.'''

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-1 Vedlegg 1 til forskriftens Kapittel 11] er Den europeiske avfallslisten (EAL). EAL angir at:

Som hovedregel skal avfall fra virksomheter m.m. som nevnt i kapittel 01 til 12 eller 17 til 20 i avfallslisten identifiseres ved hjelp av de sekssifrede avfallskodene i disse kapitlene.

Avfall som skal identifiseres ved hjelp av en avfallskode som er merket med stjerne, skal klassifiseres som farlig avfall med mindre Miljødirektoratet eller den Klima- og miljødepartementet bemyndiger har bestemt noe annet i medhold av § 11-2 tredje ledd.

[https://lovdata.no/dokument/SF/forskrift/2004-06-01-930/KAPITTEL_12#KAPITTEL_12-2 Vedlegg 2 til forskriftens Kapittel 11]angir Kriterier som gjør avfall farlig.

Punkt 1 av Vedlegg 2, Egenskaper som gjør avfall farlig, lyder som følger:

Ved vurderingen av om avfall som nevnt i § 11-2 bokstav b og vedlegg 1 nr. 3 annet ledd bokstav a skal klassifiseres som farlig, gjelder [https://lovdata.no/static/SF/32014r1357u.pdf?timestamp=1465900115000 vedlegg III til direktiv 2008/98/EF] ([http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32014R1357&from=EN engelsk versjon]) som endret ved forordning (EU) nr. 1357/2014, med unntak for HP 1, HP 9 og HP 15.(...)

'''Farlige stoffer:'''

Begrepet "farlige stoffer" brukes i forskriften. Hva er farlige stoffer? I henhold til forskriftens §11-3 er farlige stoffer definert som

''stoffer som skal klassifiseres som farlige i henhold til forordning (EF) nr. 1272/2008 artikkel 3, jf. forskrift 16. juni 2012 nr. 622 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP).''

I henhold til forordning (EF) nr 1272/2008 artikkel 3 er farlige stoffer definert som

''A substance or a mixture fulfilling the criteria relating to physical
hazards, health hazards or environmental hazards, laid down in
Parts 2 to 5 of Annex I is hazardous and shall be classified in
relation to the respective hazard classes provided for in that
Annex.''

'''Håndtering og levering av farlig avfall:'''

'''Det er ikke tillatt å fortynne farlig avfall for å komme under grenseverdiene, jf. DIRECTIVE 2008/98/EC artikkel 7 punkt 4.'''

Avfallsforskriftens § 11-5 først og andre ledd lyder som følger:

''Farlig avfall skal tas hånd om på en forsvarlig måte. Alle som oppbevarer, transporterer eller håndterer farlig avfall, skal treffe nødvendige tiltak for å unngå fare for forurensning eller skade på mennesker eller dyr.

Farlig avfall skal ikke blandes sammen med annet avfall. Ulike typer farlig avfall skal ikke sammenblandes dersom dette kan medføre fare for forurensning, eller skape problemer for den videre håndteringen av avfallet.''

Forskriftens §11-8 første ledd lyder som følger:

''Virksomhet hvor det oppstår farlig avfall, skal levere dette til den som etter § 11-6 og § 11-7 kan håndtere avfallet, eller til virksomhet utenfor Norge i henhold til reglene om grensekryssende transport av avfall i kapittel 13. Det farlige avfallet skal leveres minst 1 gang pr. år. Plikten inntrer ikke før den totale mengden farlig avfall overstiger 1 kg.''

Forskriftens §11-12 første punktum lyder som følger:

''Virksomhet som leverer farlig avfall skal gi tilstrekkelige opplysninger om avfallets opprinnelse, innhold og egenskaper, slik at den videre håndteringen av avfallet kan skje på en forsvarlig måte.''

Se også http://www.miljodirektoratet.no/no/Tjenester-og-verktoy/Veileder/Klassifisering-av-farlig-avfall-basert-pa-innhold-av-farlige-stoffer/

Se også https://www.mn.uio.no/kjemi/forskning/grupper/skole/ressurser/hms/kjemikalier-grunnskole/kjemikalier-ungdomstrinn/avfallsbehandling.pdf

'''Noen avfallskategorier som kan tenkes å være aktuelle:'''

*06 02 04 natrium- og kaliumhydroksid

*06 02 05 andre baser

*06 01 06 andre syrer

*20 01 13 løsemidler

*20 01 14 syrer

*20 01 15 baser

==Aktuelle kjemikalier==

===Tris===

http://echa.europa.eu/substance-information/-/substanceinfo/100.000.969

Faresetninger: H315, H319, H335

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H315 & H319: 20%

H335: 20%

===Eddiksyre===

Faresetninger: H226, H314

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H226: N/A. Brannfarlighet må vurderes i hvert enkelt tilfelle.

H314: For farlig avfall klasse HP4 - 1%; For farlig avfall klasse HP8 - 5%

(Når avfall inneholder ett eller flere stoffer klassifisert som Skin corr. 1A, 1B eller 1C (H314) og summen av konsentrasjonene er høyere enn eller lik 5
%, skal avfallet klassifiseres som farlig avfall av typen HP 8.)

===Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA)===

Faresetninger: H332, H373, H302, H315, H319,

Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014 :

H332: 22,5%

H373: 10%

H302: 25%

H315 & H319: 20%

Minste grenseverdi: 10%

===DMSO===

===Guanidinium chloride===

Finnes i mange kommersielle kit for DNA-rensing. Typisk konsentrasjon kan være 5 M: http://openwetware.org/wiki/Qiagen_Buffers

http://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/52449

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H302: 25%
*H315: 20%
*H319: 20%

Minste grenseverdi: 20%

Basert på dette vil en 5M løsning sannsynligvis regnes som farlig avfall.

===Isopropanol===

https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/22308

Faresetninger (Konsentrasjonsgrenseverdier for klassifisering som farlig avfall iht. COMMISSION REGULATION (EU) No 1357/2014):
*H225: " If the presence of [the] substance indicates that the waste is flammable, it shall be classified as hazardous by HP 3"
*H319 (20%)
*H336 (no limit given)

==Aktuelle stoffblandinger==

===TAE buffer===

Blanding av Tris-Acetate-EDTA. Se aktuelle kjemikalier for grenseverdier for de enkelte stoffene til bruk ved vurdering av klassifisering som farlig avfall. Typiske konsentrasjoner i 50x konsentret buffer kan være:

Tris: ~25%
Eddiksyre: ´~6 %.
EDTA: <1%.

Jf. begrensende grenseverdier for Tris, Eddiksyre og EDTA basert på disses faresetninger og lik henholdsvis 20%, 1% og 10% vil innholdet av Tris og eddiksyre dermed sannsynligvis medføre at konsentrert, ufortynnet 50X TAE buffer må klassifiseres som farlig avfall ved avhending. Se Aktuelle kjemikalier.

For 10X TAE buffer vil de aktuelle konsentrasjonene være ca.:

Tris: ~5%
Eddiksyre: 1,2%
EDTA: <0.2%

På grunn av innholdet av eddiksyre vil dermed også 10X TAE buffer sannynligvis klassifiseres som farlig avfall ved avhending.

Konsentrert buffer fortynnes typisk til 1x før bruk. Brukt buffer vil dermed sannsynligvis ikke inneholde konsentrasjoner av de nevnte stoffene over grenseverdiene, og kan i såfall avhendes som vanlig avfall. (OBS: Det er ikke tillatt å fortynne farlig avfall med den hensikt å komme under grenseverdiene).

'''Påkrevd piktogram iht. CLP: GHS07'''

==Sikkerhetsdatablader==

EU-krav til sikkerhetsdatablader er gitt i Annex II til REACH: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

Se også EHCA Guidance on the compilation of safety data sheets: http://echa.europa.eu/documents/10162/13643/sds_en.pdf

GelGreen: http://biotium.com/wp-content/uploads/2013/07/MSDS-41005.pdf

DSView Nucleic acid stain: https://drive.google.com/open?id=0B9aq85qBYTsWbUJCYTZNTzNHSFFTS1BvdzB0bWZ0NTZjby04

DSBio Taq mix (2x) P2011,P2012: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

DSBio 50 bp ladder: https://drive.google.com/open?id=0B9aq85qBYTsWSDlxR2Vra1ZiUUdFRlpNeFduSHl2TzZnUC1Z

Notis: Inneholder 1-5% 2-amino-2-(hydroxymethyl)-1,3-propanediol, faresetninger
Xi, R 36/37/38. (H319/H335/H315) (irriterende). '''Ufortynnet produkt klassifiseres derfor som farlig avfall klasse HP4.''', jf. Annex III to Directive 2008/98/EC som revidert ved EU-direktiv 1357/2014

DSBio gel loading dye: https://drive.google.com/open?id=0B9aq85qBYTsWRWk3Qk5jVmJfWlc0NUswTVU4eGVLbk9rS2xB

DSBio NF water: https://drive.google.com/open?id=0B9aq85qBYTsWS1JlUS03V19WUHJGY21OWFVmTHhYSmhIQXVj

National Diagnostics TAE 50x: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-872

Merck Millipore agarose for elektroforese: http://www.merckmillipore.com/INTERSHOP/web/WFS/Merck-INTL-Site/en_US/-/USD/ShowDocument-File?ProductSKU=MDA_CHEM-116802&DocumentType=MSD&Language=NO&Country=NO

==Faremerking==

https://www.pervaco.no/skilt-fundament/ghs-clp-skilt

http://www.miljodirektoratet.no/no/Publikasjoner/Publikasjoner/2011/Mars/Klassifisering_og_merking_i_CLP/

http://www.erdetfarlig.no/no/Artikler/faremerking/?PageID=74

Konverter fra r-setninger til H-setninger: http://ghs.dhigroup.com/PagesGHS/TranslationTool.aspx

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Om-klassifisering-og-merking-av-kjemikalier-CLP/

Kjemisk sikkerhet og din virksomhet - informasjonsplakat fra miljødirektoratet: http://www.miljodirektoratet.no/Documents/publikasjoner/M324/M324.pdf

Innføring i klassifisering og merking av kjemikalier: http://www.miljodirektoratet.no/Documents/publikasjoner/M247/M247.pdf

Plakat med oversikt over klassifisering og merking etter CLP: http://www.miljodirektoratet.no/Documents/publikasjoner/M259/M259.pdf

==Databaser==

http://echa.europa.eu/information-on-chemicals

http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database

=Lenker=

==Lab techniques==

Alkaline lysis: http://bitesizebio.com/180/the-basics-how-alkaline-lysis-works/

==Andre grupper og nettsteder==

http://www.socializedscience.com/projects1.html

BioHack Academy: https://biohackacademy.github.io/

https://diybio.org/

https://www.facebook.com/groups/diybio/

GenSpace (New York): http://genspace.org/

La paillasse (PAris) http://lapaillasse.org/

http://biocurious.org/

Biologigaragen (København): http://biologigaragen.org/ & https://www.facebook.com/groups/biologigaragen/

https://biohackspace.org/

http://www.diybiogroningen.org/

http://www.indiebiotech.com/

Liste over grupper på DIYbio.org: https://diybio.org/local/

http://biohackingsafari.com/

BioHacklabs.org Wiki: http://www.biohacklabs.org/Main_Page

Robert Carlson: http://synthesis.cc

London Biohackspace: http://biohackspace.org/

==HMS==

WHO Laboratory Biosafety Manual, Third Edition: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Biosafety in Microbiological and Biomedical Laboratories
5th Edition, U.S. Department of Health and Human Services: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf

NTNU - Arbeid med biologiske faktorer: https://innsida.ntnu.no/wiki/-/wiki/Norsk/Arbeid+med+biologiske+faktorer

https://innsida.ntnu.no/wiki/-/wiki/Norsk/Biologiske+faktorer

http://www.reach-chemconsult.com/en/seiten/ghs-konverter.html

http://www.the-scientist.com/?articles.view/articleNo/14477/title/An-Accident-Waiting-to-Happen-/

'''Elektroforese:'''

https://web.stanford.edu/dept/EHS/prod/researchlab/lab/safety_sheets/08-136.pdf

http://www.ab.ust.hk/hseo/tips/ls/ls008.htm

http://ehs.unl.edu/sop/s-electrophoresis_safety.pdf

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.labmanager.com/lab-health-and-safety/2010/05/electrophoresis-safety-tips?fw1pk=2#.V2Wh3_mLRD8

https://www.admin.ox.ac.uk/safety/policy-statements/s11-07/

http://www.di.uq.edu.au/sparq/RAs/SPARQedDNARNAElectrophoresisRA.pdf

==Lover og forskrifter==

Lover og forskrifter som er lenket til vil ikke nødvendigvis gjelde for aktiviteter ved Bitraf, men kan likevel brukes som en kilde til HMS-relevant informasjon og veiledning:

===Norske forskrifter===

Forskrift om særavgifter (relevant mht. bruk av teknisk sprit): http://lovdata.no/forskrift/2001-12-11-1451/§3-3-10

Forskrift om utforming og innretning av arbeidsplasser og arbeidslokaler (arbeidsplassforskriften) - Kapittel 8. Arbeid i omgivelser som kan medføre eksponering for biologiske faktorer: http://lovdata.no/forskrift/2011-12-06-1356/§8-1

Forskrift om tiltaksverdier og grenseverdier for fysiske og kjemiske faktorer i arbeidsmiljøet samt smitterisikogrupper for biologiske faktorer (forskrift om tiltaks- og grenseverdier) : https://lovdata.no/dokument/SF/forskrift/2011-12-06-1358

Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav (forskrift om utførelse av arbeid) -Andre del: Krav til arbeid med kjemiske og biologiske risikofaktorer: https://lovdata.no/dokument/SF/forskrift/2011-12-06-1357/KAPITTEL_2#KAPITTEL_2

Forskrift om begrensning i bruk av helse- og miljøfarlige kjemikalier og andre produkter (produktforskriften): https://lovdata.no/dokument/SF/forskrift/2004-06-01-922#KAPITTEL_1

Merk spesielt §5-1:

§ 5-1.Omsetning og import av meget giftige og giftige kjemikalier til privat bruk
Til privat bruk er det forbudt å importere kjemikalier merket med faresymbol og farebetegnelse «meget giftig» eller «giftig» i henhold til forskrift om klassifisering, merking mv. av farlige kjemikalier eller som i henhold til forordning (EF) nr. 1272/2008 om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP-forordningen) skal klassifiseres i fareklasse og farekategori Carc. 1A, Carc. 1B, Muta. 1A, Muta. 1B, Repr. 1A, Repr. 1B, Acute Tox. 1, Acute Tox. 2, Acute Tox. 3, STOT RE 1 eller STOT SE 1. Forbudet mot privat import gjelder ikke for motorbensin eller dieselolje til transportformål som innføres på kjøretøyets drivstofftank eller i godkjente reservetanker

https://lovdata.no/dokument/SF/forskrift/2004-06-01-930

===REACH & CLP===

1272/2008 EC (CLP): http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF

Forskrift om klassifisering, merking og emballering av stoffer og stoffblandinger (CLP): https://lovdata.no/dokument/SF/forskrift/2012-06-16-622

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Virkeomrade-CLP-avsnitt-I/

Forholdet mellom REACH og CLP:

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Klassifisering-og-merking-av-kjemikalier-CLP/Forholdet-mellom-REACH-og-CLP/

http://www.miljodirektoratet.no/no/Tema/Kjemikalier/Kjemikalieregelverk/Kjemikalieregelverket_REACH/Unntak/

http://echa.europa.eu/addressing-chemicals-of-concern/restrictions/substances-restricted-under-reach

Offisiell tekst: http://eur-lex.europa.eu/legal-content/en/TXT/PDF/?uri=CELEX:02006R1907-20160401

REACH angir plikter både for produsenter/importører og for "downstream users". "Downstream users" er definert som

"companies or individuals:

within the European Union/European Economic Area,
who use a substance, either on its own or in a mixture,
in their industrial or professional activities."

Antar at våre aktiviteter ikke vil omfattes av denne definisjonen?

Se også http://echa.europa.eu/regulations/reach/downstream-users og http://www.prc.cnrs-gif.fr/reach/en/user_obligations.html

ECHA Navigator: http://echa.europa.eu/support/guidance-on-reach-and-clp-implementation/identify-your-obligations/navigator

http://www.steptoe.com/assets/htmldocuments/Atlee_-_Downstream_Users.pdf

http://echa.europa.eu/documents/10162/966058/tips_users_chemicals_workplace_en.pdf

REACH database: http://echa.europa.eu/information-on-chemicals/registered-substances

==Litteratur==

===Bøker===

Biohackers: The Politics of Open Science: http://www.amazon.com/Biohackers-Politics-Science-Alessandro-Delfanti/dp/0745332803/ref=sr_1_3?s=books&ie=UTF8&qid=1463928520&sr=1-3&keywords=biohacker

Open-Source Lab: How to Build Your Own Hardware and Reduce Research Costs: http://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DXhttp://www.amazon.com/dp/0124104622/ref=wl_it_dp_o_pC_S_ttl?_encoding=UTF8&colid=2JXTKSS1LI8NT&coliid=I2AIVHRBOC69DX

Biopunk: Solving Biotech's Biggest Problems in Kitchens and Garages: http://www.amazon.com/Biopunk-Solving-Biotechs-Problems-Kitchens/dp/1617230073/ref=sr_1_1?s=books&ie=UTF8&qid=1463930140&sr=1-1&keywords=biopunk

The Machinery of Life: http://www.amazon.com/Machinery-Life-David-S-Goodsell/dp/0387849246/ref=pd_sim_14_5?ie=UTF8&dpID=51ZSNcQ3vrL&dpSrc=sims&preST=_AC_UL160_SR106%2C160_&refRID=156THN5QQ1RD2Q4DQ5XB

Illustrated Guide to Home Biology Experiments: http://www.amazon.com/Illustrated-Guide-Home-Biology-Experiments/dp/1449396593?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o08_s00

Techniques in microbiology - a student handbook: http://www.amazon.com/Techniques-Microbiology-Handbook-John-Lammert/dp/0132240114?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o04_s00

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life: http://www.amazon.com/dp/0674060156/ref=rdr_ext_tmb

Exploring Personal Genomics: http://www.amazon.com/Exploring-Personal-Genomics-Joel-Dudley/dp/0199644497?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

The Art of Fermentation: An In-Depth Exploration of Essential Concepts and Processes from around the World: http://www.amazon.com/Art-Fermentation-Depth-Exploration-Essential/dp/160358286X?ie=UTF8&psc=1&redirect=true&ref_=oh_aui_detailpage_o06_s00

===Artikler===

'''DIY Bio:'''

European do-it-yourself (DIY) biology: Beyond the hope, hype and horror: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4158858/

DIYBIOLOGISTS AS ‘MAKERS’ OF PERSONAL BIOLOGIES: HOW MAKE MAGAZINE AND MAKER FAIRES CONTRIBUTE IN CONSTITUTING BIOLOGY AS A PERSONAL TECHNOLOGY: http://peerproduction.net/issues/issue-2/peer-reviewed-papers/diybiologists-as-makers/?format=pdf

http://blogs.plos.org/synbio/2016/05/03/synbio-democratizing-biotechnology/

Synthetic biology: from mainstream to counterculture.: http://www.ncbi.nlm.nih.gov/pubmed/27316777

'''DNA barcoding:'''

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066213

Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for
Fungi:
https://www.academia.edu/12648759/Nuclear_ribosomal_internal_transcribed_spacer_ITS_region_as_a_universal_DNA_barcode_marker_for_Fungi?auto=view&campaign=weekly_digest

'''Hardware:'''

Edwin: A Robotic Platform for Automated RNA Extraction and Analysis during Reporter Gene–Based Dynamic Characterization of Bacterial Promoters: http://jla.sagepub.com/content/early/2016/06/17/2211068216655151.long

===Nyheter===

http://www.prnewswire.com/news-releases/genspace-nyc-receives-350000-in-support-from-the-simons-foundation-300237457.html

==Kommersielt tilgjengelig utstyr==

http://www.edvotek.com/Equipment

http://no.frederiksen.eu/

Anmeldelser, artikler, etc.: http://www.selectscience.net/

===PCR===

"MiniPCR" (Samme pris som ferdigbygd OpenPCR, $650. Ikke DIY): http://www.minipcr.com/product-category/equipment-and-accessories/minipcr-thermal-cycler/

OpenPCR: http://openpcr.org/

===Elektroforese og transilluminasjon===

DarkReader Blue Light Transilluminator: http://www.clarechemical.com/transilluminator.htm

===Spektro-/foto-/fluorometri===

Qubit 3.0 fluorometer:https://www.thermofisher.com/order/catalog/product/Q33216. Listepris 15 340,00 NOK.

Spektrofotometer V1100D: http://no.frederiksen.eu/nettbutikk/felles-laboratorieutstyr/maaleutstyr/spektrofotometer-v1100d. Listepris 5 995,00 kr eks. MVA.

http://www.ebay.com/itm/Visible-Spectrometer-Laboratory-Spectrophotometer-220V-350-1020nm-721-/401130883021?hash=item5d654387cd:g:ONoAAOSwMmBVoPei. Listepris ca. NOK 2,329.51.

===Annet laboratorieutstyr===

http://no.frederiksen.eu/shop/product/termostatblokk--labnet--digital--enkel

http://no.frederiksen.eu/shop/product/bakteriedyrkingsskap-cultura

==Kommersielt tilgjengelige reagenser og forbruksvarer==

===Mikrobiologi===

Peptone LP0037: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0037

Oxoid yeast extract: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=LP0021&c=UK&lang=EN

===PCR===

http://www.minipcr.com/product-category/minipcr-learning-labs-and-kits/

===Elektroforese og transilluminasjon===

https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/

http://pearlbiotech.com/

===Restriksjonsenzymer===

EcoRI:

Gjenkjenningssekvens (plusstråd/komplementær): GAATTC / GAATTC

*http://no.frederiksen.eu/shop/product/restriksjonsenzym-ecori
*https://www.neb.com/products/r0101-ecori

CfoI:

Gjenkjenningssekvens (plusstråd/komplementær): GCGC / GCGC

*https://no.promega.com/products/cloning-and-dna-markers/restriction-enzymes/cfoi/

HaeIII:

Gjenkjenningssekvens (plusstråd/komplementær): GGCC / GGCC

*https://www.neb.com/products/r0108-haeiii

HinfI:

Gjenkjenningssekvens (plusstråd/komplementær): GANTC/GANTC

https://www.neb.com/products/r0155-hinfi

Relevant litteratur:

Clark et al. Extended stability of restriction enzymes at ambient temperatures. Biotechniques. 2000 Sep;29(3):536-8, 540, 542.: http://www.ncbi.nlm.nih.gov/pubmed/10997268

==Leverandører av utstyr, tjenester og forbruksvarer==

===Forbruksvarer og reagenser===

http://www.metabion.com/products/index.php

http://www.the-odin.com/

http://www.geneandcell.com/

http://dongshengbio.com/en/index.asp

http://www.onlinesciencemall.com/ (Selger bl.a. TAE buffer)

http://www.abpbio.com/product/nucleic-acid-solution-quantitation-kits/

https://zageno.com/

===Oligomersyntese===

http://macrogen.com/eng/

http://www.sigmaaldrich.com/technical-documents/articles/biology/standard-dna-synthesis.html

http://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html

===Sekvensering===

'''General advice:'''

http://www.bgi.com/services/genomics/sanger-sequencing/single-sample-sequencing/

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-choosing.html

http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

'''GATC:'''

LightRun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/lightrun-sequencing.html'

*Prepaid labels
*Premixed DNA and primer
*4.00 EUR / reaction, minimum order of 100
*(Welcome offer: 50 reactions for 3.50 EUR each)

SUPREMErun sequencing: https://www.gatc-biotech.com/en/products/sanger-services/supremerun-sequencing.html

'''Macrogen:'''

http://macrogen.com/eng/

http://dna.macrogen.com/eng/support/ces/guide/order_guide.jsp

Standard sequencing: https://dna.macrogen.com/eng/order/ces/std/s_new_step1.jsp
*5.99 EUR/reaction (express, 24 turnaround)
* 4.5 EUR/reaction (regular, 4-5 workdays turnaround)
*+2 EUR/reaction for purification

EZseq: http://dna.macrogen.com/eng/order/ces/ezseq/ezseq_step1.jsp

*EZseq single direct: 4.5 EUR/label (minimum order of 50).
*24hr turnaround

Sample purification is not offered with EZseq.

Eco-seq: http://dna.macrogen.com/eng/order/ces/ecoseq/ecoseq_step1.jsp

*Eco-seq single direct: 4.99 EUR/label (minimum order of 50)
*Eco-seq single purification: 6.99 EUR/label

Custom sequencing: https://dna.macrogen.com/eng/support/ces/customized_seq_intro.jsp

Sample preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

From FAQ: "Minimum 20ul of 100ng/ul(plasmids, unpurified PCR products) or 50ng/ul(purified PCR products) are required for a couple of reactions."

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

'''BaseClear:'''

See http://www.baseclear.com/genomics/sanger-sequencing

See https://orders.baseclear.com/

Single run sequencing services:

Prepaid barcode sequencing: http://www.baseclear.com/genomics/sanger-sequencing/prepaid-barcode-sequencing
*DNA purification and premixing with primer is done by the customer

Quickshot: http://www.baseclear.com/genomics/sanger-sequencing/quick-shot

Primers options:
*Separate sample and primer or premix possible
*Free usage of universal primers (see standard primer list)
*Custom primer, sent with the order (10 pmol/uL in volume >20 uL (enough for max.10 reactions)

Sample options:
*Bacteria for plasmid isolation (miniprep) on a agar plate or as glycerol stock
*Purified plasmid in a minimal volume of 30 ul with a DNA concentration of 50 – 200 ng/ul
*Purified or Raw PCR product in a minimal volume of 30 ul with a DNA concentration >5 ng/μl

Pricing: ??
Payment: Invoice by e-mail.

'''Source BioScience:'''

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/

http://www.lifesciences.sourcebioscience.com/genomic-services/sanger-sequencing-service/information/sample-requirements/

9.5 EUR/reaction for minimum order of 50 (475 EUR)

Concentration requirement for (purified) PCR product: 1ng/µl per 100bp

*Payment by credit card available.

==DIY/Open hardware==

http://openwetware.org/wiki/DIYbio:Notebook/Open_Gel_Box_2.0

https://www.bento.bio/

https://www.chaibio.com/

http://hackteria.org/wiki/index.php/DIY_NanoDrop

http://www.thingiverse.com/thing:73910

==Prosjekter til inspirasjon==

http://www.instructables.com/id/DIY-Bio-plastics/

==Diverse==

http://dna-view.com/

=Bioinformatikk=

==Genomikk==

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/info/definitions.shtml

==Databaser==

[http://www.ncbi.nlm.nih.gov/genbank/ GenBank]

RefSeq: http://www.ncbi.nlm.nih.gov/refseq/about/

http://www.yeastgenome.org/

http://www.ncbi.nlm.nih.gov/refseq/

Om NCBI Genome Assembly model: http://www.ncbi.nlm.nih.gov/assembly/model/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702866/

==Referansesekvenser==

===Gjær===

'''Saccharomyces cerevisiae:'''

S288C: http://www.yeastgenome.org/strain/S288C/overview#resources

http://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.g3journal.org/content/4/3/389.full

'''Brettanomyces (Dekkera) bruxellensis:'''

http://www.ncbi.nlm.nih.gov/genome/11901

http://www.ncbi.nlm.nih.gov/assembly/GCA_000340765.1

http://www.ncbi.nlm.nih.gov/pubmed/22663979

==Søkeverktøy==

[https://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI BLAST]

==Nedlastbare programmer==

[http://www.sanger.ac.uk/science/tools/artemis Artemis]

==Andre verktøy==

In silico PCR: https://genome.ucsc.edu/cgi-bin/hgPcr

http://www.complex.iastate.edu/download/Picky/index.html

=Biobanker/artskataloger=

Spanish Type Culture collection (CECT): http://www.cect.org/english/hongos.php

=Organismer=

==Gjær==

===Saccharomyces===

http://www.klikk.no/mat/spise/article1490066.ece

http://wiki.yeastgenome.org/index.php/What_are_yeast%3F

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962479/

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now: http://www.straininfo.net/strains/317495

http://www.ncbi.nlm.nih.gov/genome/?term=txid4932[orgn]

https://www.phys.ksu.edu/gene/chapters.html

http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

5.8S rRNA: http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017404&trail=|1017404

[http://www.yeastgenome.org/browse/?loc=chrXII%3A455382..455603&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight= 5.8S rRNA (RDN58-2) at SGD] (Lokasjon chrXII:455414..455571)

5.8S rRNA (RDN58-1) at SgD:http://yeastmine.yeastgenome.org/yeastmine/report.do?id=1017401&trail=|1017401 (Lokasjon: chrXII:455414-455571 reverse strand)

Chromosome XII context is important for rDNA function in yeast: http://nar.oxfordjournals.org/content/34/10/2914.full

===Brettanomyces===

NCBI organism page: http://www.ncbi.nlm.nih.gov/genome/?term=txid5007[orgn]

Crauwels et al. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing. Appl Environ Microbiol. 2014 Jul; 80(14): 4398–4413. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068659/

Wikipedia: https://en.wikipedia.org/wiki/Brettanomyces_bruxellensis

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis. http://femsle.oxfordjournals.org/content/284/2/213

https://lup.lub.lu.se/student-papers/search/publication/3632990

==Mesoplasma florum==

http://www.ncbi.nlm.nih.gov/genome/?term=Mesoplasma+florum

=Protokoller=

Se https://bitraf.no/wiki/BioHackerLab/Protokoller

BioHackerLab/Protokoller

2016-08-07T23:15:16Z

Jarlemag:

=Resuspendering av DNA-primere=

Ta røret med tørket DNA ut av pakken. Kontroller at ID og DNA-sekvens oppgitt på røret stemmer med bestillingen.

Plasser røret i en mikrosentrifuge og sentrifuger i ca. 30 sekunder.

Tilsett NF-vann til konsentrasjon etter oppløsning lik 100 pmol/uL (0.1 mM, 100 uM). Se siden av røret eller medfølgende dokumentasjon fra leverandør for total mengde DNA i røret og/eller mengde vann som må tilsettes for endelig konsentrasjon ~100 pmol/uL

Bland ved å knipse på og riste flasken, eller med en vortex-mikser. Sentrifuger til slutt røret igjen.

Oppbevar fryst, fortrinnsvis ved -20C.

Før bruk til PCR vil det være hensiktsmessig å foreta ytterligere 10X fortynning til 10 pmol/uL (10 uM) i et eget rør, som også kan oppbevares fryst og brukes flere ganger. Dette reduserer også behovet for å åpne hovedrøret og reduserer risikoen for kontaminasjon av dette.

Se også https://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html?

Om mulig kontroller konsentrasjonen vha. spektrofotometri. Se http://www.promega.com/a/apps/biomath/index.html?calc=odConvert

=DNA extraction=

Blount, Driessen & Ellis: GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA. Scientific Reports 6, Article number: 26863 (2016). http://www.nature.com/articles/srep26863

Strawberry crude DNA extraction: http://www.stevespanglerscience.com/lab/experiments/strawberry-dna/

=DNA quantification=

==Background==

The main methods for determiniing nucleic acid concentrations are UV photometry measuring absorbance at 260 nm (A260) and fluorometry. Note that different sources may describe different conversion factors for conversion from A260 to mass concentration. For oligomers, a more accurate DNA concentration value may be achieved by estimating the sequence-dependent extinction coefficient by calculation.

https://www.scripps.edu/california/research/dna-protein-research/forms/biopolymercalc2.html

http://biotools.nubic.northwestern.edu/OligoCalc.html

http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full

http://www.promega.com/a/apps/biomath/

https://no.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/

http://www.ogt.co.uk/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality

https://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/09%20UV%20Absorption.pdf

https://www.idtdna.com/pages/support/technical-vault/reading-room/quick-reference/quick-reference/2011/06/02/molar-extinction-coefficient

http://www.endmemo.com/bio/OD260.php

=Elektroforese=

Se også: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_SeaKem_LE_Agarose_-_Protocol.pdf

'''OBS: Les gjennom hele protokollen før gjennomførelse for å bli kjent med relevant sikkerhetsinformasjon.'''

==Utstyr==

'''OBS: Kontroller utstyret for skader og slitasje før bruk. Ikke fortsett dersom elektroforesekammer, ledninger eller strømforsyning viser tegn til skade.'''

*Elektroforesekammer. Eks. Carolina Deluxe Gel Chamber.
*Strømforsyning. Eks. BioRad PowerPac Basic
*Automatpipette, 1-10 uL.
*Vekt, 0.1 g eller høyere oppløsning.
*Mikrobølgeovn
*Varmebeskyttende hansker eller annen håndbeskyttelse
*Målesylinder, 50 mL eller større
*Glassflaske til agaroseløsning, 100 mL eller større (må passe i mikrobølgeovn)
*Glassflaske til TAE-bufferløsning, 250 mL eller større
*Transilluminator, eks. DarkReader blue light.
*Stekespade eller lignende for å løfte agarosegel

Avfallsbeholder for pipettespisser og tørt avfall
Avfallsbeholder for brukt buffer/flytende avfall

Forbruksvarer:
*Agarose for elektroforese, eks. SeaKem LE agarose. Ca. 0.5 g/gel.
*TAE 10x buffer. Ca. 30 mL.
*Destillert vann: Ca. 300 mL.
*DNA-fargestoff, eks. GelGreen. Ca 5 uL.
*Pipettespisser, 1-10 uL.
*Veieskip.
*Tørkepapir
*DNA ladder
*Loading dye

==Støping av agarose-gel==

Om nødvendig, lag TAE buffer i brukskonsentrasjon ved å fortynne konsentrert buffer i destillert vann. For å lage 1x TAE buffer fra 10x TAE buffer, bland 1 del TAE buffer og 9 deler destillert vann. TAE buffer er irriterende. Bruk hansker ved håndtering av konsentrert buffer.

Tilsett gradvis ~1 v/v % agarose til 1x TAE buffer (~1g til 100 mL, f.eks 0.5g for 50 mL) i en flaske med volum minst to ganger volumet av løsningen, fortrinnsvis under omrøring med magnetrører - eventuelt rør for hånd med glasstav eller ved å bevege flasken.

'''OBS: Flasken må ikke være lufttett!'''

Varm løsningen i mikrobølgeovn på høy styrke til løsningen koker (ca. ett minutt). La løsningen koke i ca. 30 sekunder. Reduser varmeeffekten eller skru av ovnen dersom løsningen koker over.

Fjern flasken fra mikrobølgeovnen. Bland løsningen ved å bevege flasken '''forsiktig''' i en roterende bevegelse for hånd, eller med en magnetrører. Utvis forsiktighet for å unngå sprut. Kontroller at agarose-løsningen er klar og uten synlige partikler. Dersom det er partikler og uløst agarose i løsningen, kok løsningen igjen.

'''OBS: Bruk øyebeskyttelse! Fare for støtkoking og sprut også etter at oppvarmingen har opphørt. Flasken er varm. Bruk varmebeskyttende hansker eller annen håndbeskyttelse ved håndtering av flaske. Bruk fortrinnsvis labfrakk eller langarmede klær og unngå eksponering av bar hud. Fare for forbrenning ved søl eller sprut.'''

Tilsett DNA-fargestoff i henhold til konsentrasjonsangivelse. F.eks, for 10 000x konsentrert fargestoff, tilsett 1 uL fargestoff per 10 mL agarose løsning (5 uL for 50 mL).

Plasser et gel-støpekar på tvers av lengderetningen i et elektroforesekar og plasser en brønnkam i støpekaret. Hell varm agaroseløsning i kammeret og la stå til gel'en er størknet (ca. 30-60 minutter).

'''OBS: Elektroforesekaret skal være frakoblet fra strømforsyning når gel'en støpes.'''

Ta ut brønnkammen og snu støpekaret slik at prøvebrønnene er nærmest den sorte (postive) elektroden.

Hell 1x TAE buffer i elektroforesekaret slik at gel'en er dekket av buffer.

==Elektroforese==

*Tilsett prøvene i prøvebrønnene.
*Sett på lokket på elektroforesekaret.
*Kontroller at området rundt elektroforesekaret og strømforsyningen er tørt. Tørk bort eventuell væske.
*Koble ledningene til strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.

'''OBS: Strømforsyningen skal være avslått når ledningene kobles til!'''

*Skru på strømforsyningen.
*Juster til ønsket spenning og skru på spenningen.
*La elektroforesen foregå uforstyrret. '''Ikke rør kammeret eller ledningene så lenge spenningen er på. Dersom det oppstår tegn til lekkasje fra kammeret, avslutt elektroforesen umiddelbart uten å komme i kontakt med væsken.'''
*Skru av spenningen på strømforsyningen.
*Skru av strømforsyningen.
*Koble ledningene fra strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.
*Ta av lokket på elektroforesekaret.
*FLytt agarosegeleen forsiktig til en transilluminator ved hjelp av en stekespade e.l.
*Avhend agarose-gel, bufferløsning og forbruksmateriell på forsvarlig vis etter bruk. Skyll elektroforesekammer og glassutstyr med destillert vann.

Se også: https://www.addgene.org/plasmid-protocols/gel-electrophoresis/

http://www.methodbook.net/dna/agarogel.html

Typisk prøvevolum kan være 6 uL, f.eks 5 uL PCR-produkt + 1 uL 6x loading dye, eller 4 uL vann, 1 uL DNA ladder og 1 uL 6x loading dye.

=PCR=

Typiske reaksjonsvolum er 20 uL og 50 uL. Gitt en konsentrasjon i primerløsningen lik 10 uM vil i de to tilfellene tilsetting av henholdsvis 1 uL og 2.5 uL av hver primer gi en endelig kosentrasjon av hver primer lik 0.5 uM i reaksjonsblandingen.

http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Typisk vil en ta ut ca. 5 uL av reaksjonsblandingen til elektroforese-analyse etter PCR.

Se også https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

==Detection of D1S80 Repeat Polymorphism by PCR==

See https://www.dnalc.org/files/pdf/forensicprofchip_d1s80_protocol.pdf

==Collection of genomic DNA by buccal swabs==

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566681/pdf/envhper00512-0045.pdf

==PTC tasting ability genotyping==

https://www.snpedia.com/index.php/Rs713598

https://www.ncbi.nlm.nih.gov/pubmed/12595690?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181941/

http://www.ncbi.nlm.nih.gov/gene?cmd=retrieve&dopt=default&rn=1&list_uids=5726

http://bioinformatics.dnalc.org/ptc/animation/pdf/ptc.pdf

==Sex determination by PCR==

See https://en.wikipedia.org/wiki/Amelogenin#Application_in_sex_determination and Fracnes et al.: Clin Chim Acta. 2007 Nov-Dec;386(1-2):53-6. Epub 2007 Jul 31.
Amelogenin test: From forensics to quality control in clinical and biochemical genomics.. (http://www.sciencedirect.com/science/article/pii/S0009898107003889)

Primers:

*AME-F: 5'-ctgatggttggcctcaagcctgtg-3'
*AME-R: 5'-taaagagattcattaacttgactg-3'

Expected results:

*If male: 1 band of 977 bp + 1 band of 790 bp
*If female: 1 band of 977 bp

==DNA fingerprinting==

===CODIS===

The Combined DNA Index System (CODIS) is a FBI program to support law enforcement by DNA-based identification. CODIS defines standards for DNA fingerprinting and a selection of genetic loci to be analyzed as part of DNA fingerprinting. The 13 CODIS core loci per 2016 are:

*CSF1PO
*FGA
*THO1
*TPOX
*VWA
*D3S1358
*D5S818
*D7S820
*D8S1179
*D13S317
*D16S539
*D18S51
*D21S11

In addition, the Amelogenin (AMEL) locus is used for sex determination.

For information on each locus, see http://www.cstl.nist.gov/strbase/

See also http://www.cstl.nist.gov/strbase/coreSTRs.htm

7 additional loci will be required from January 1 2017.

http://isogg.org/wiki/CODIS

https://en.wikipedia.org/wiki/Combined_DNA_Index_System

http://www.cstl.nist.gov/biotech/strbase/fbicore.htm

http://www.cybertory.org/resources/CODIS/

Estimated cost for CODIS primer set as described by cybertory.org, with synthesis by Macrogen Inc.: 68.2 EUR / ~75 USD / ~ 650 NOK + shipping.

===European set===

The European Standard Set (ESS) consists of the following STR loci: FGA, TH01, VWA, D1S1656, D2S441, D3S1358, D8S1179, D10S1248, D12S391, D18S51, D21S11, D22S1045

Additional Loci commonly found in European STR kits include: D2S1338, D16S539, D19S433, SE33, Amelogenin

==Colony PCR==

1. From an agar plate, select one or several colonies for colony PCR. For each colony selected, pick a small amount of colony material using a sterile pipette tip, and dissolve in 50 uL H2O. Reseed a new agar plate with 5 uL of the resulting solution(s) each in a separate spot, keeping the spots separate and noting the location of each. Alternatively, use 5 uL to inoculate a liquid culture.

Incubate the dissolved colony material at 96 C for 10 min to release DNA.

Use 1 uL of the heat-treated solution as template for PCR.

==ITS1 + ITS 4 yeast==

===Background===

See Esteve-Zarzoso et al.: ''Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers''. (http://www.ncbi.nlm.nih.gov/pubmed/10028278)

[https://www.researchgate.net/publication/13261852_Esteve-Zarzoso_B_Belloch_C_Uruburu_F_Querol_A_Identification_of_yeasts_by_RFLP_analysis_of_the_58S_rRNA_gene_and_the_two_ribosomal_internal_transcribed_spacers_Int_J_Syst_Bacteriol_49_329-337 Artikkel tilgjengelig via ResearchGate]

See also http://sites.biology.duke.edu/fungi/mycolab/primers.htm

[[Fil:ITS1 ITS4 insilico UCSC.png|miniatyr|høyre|Screenshot of in silico PCR result using the UCSC in silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr) with the primer sequences ITS1 (5' TCCGTAGGTGAACCTGCGG 3') and ITS4 (5' TCCTCCGCTTATTGATATGC 3'), selecting the S. cerevisae April 2011 (SacCer_Apr2011/sacCer3) genome assembly as the template. The result shows two expected PCR products/target regions with identical sequences. For each, the line starting with a > specifies the target location in the assembly, the target region size/expected product size, and the primer sequences, separated by spaces. The subsequent lines show the target region/expected product sequence, with the sequence of the forward primer (ITS1) and the sequence matching the reverse primer (ITS4) in capitals. The target region size/expected product size (841bp) includes the primers.]]

===PCR setup===

'''According to Esteve-Zarzoso et al.:'''

Reaction volume: 100 uL

Primer concentrations: 0.5 uM each

Forward (FWD) primer (ITS1): 5' TCCGTAGGTGAACCTGCGG 3'

Reverse (REV) primer (ITS4): 5' TCCTCCGCTTATTGATATGC 3'

(Start and end of expected product sequence = FWD primer + reverse complement of reverse primer: TCCGTAGGTGAACCTGCGG-GCATATCAATAAGCGGAGGA)

Template: Fresh yeast colony material.

Template preparation: 95 C for 15 min.

Initial denaturation: 95C for 5 min

Repeated cycles:
*Denaturation: 94 C for 1 min
*Anneal: 55.5 C for 2 min
*Extension 72C for 2 min

x 35 cycles

Final extension: 72C for 10 min

Total hold time: 190 min

Estimated program duration with OpenPCR: ~4 hr.

'''Alternative setup and reduced duration PCR program:'''

Reaction volume: 50 uL

Template: The protocol has been tested with dry yeast, see template preparation below. The protocol may also be attempted with fresh store-bought yeast, liquid yeast culture or yeast agar colony material as the template source.

PCR setup otherwise as original protocol.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 35 cycles

Step durations and temperatures are according to recommendations for Dongsheng Taq polymerase. Annealing temperature according to original protocol.

Final extension: 72 C for 10 min

Total hold time: 83 min

Run time with OpenPCR: ~ 2h 20 min.

'''Template preparation:'''

Dissolve 0.1g dry yeast (Idun tørrgjær) in 10 mL dH20. Mix well. Pipette 50 uL (-> ~0.0005g, 0.5mg yeast powder by dry weight) into a microsentrifuge tube or PCR tube and incubate at 98C for 10 minutes. Use 1-2 uL (-> ~0,00002g = 0,02mg = 20 ug yeast powder by dry weight for 2 uL) of the supernatant as template.

I tørrvekt kan det forventes at DNA utgjør i størrelsesorden 1% av cellemassen. ([http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/ ref])
20 ug tørrmasse vil da gi anslagsvis ~0.2 ug = 200 ng DNA.

DNA har molekylær vekt lik ca. 650 g/mol per basepar. ([https://www.neb.com/tools-and-resources/usage-guidelines/nucleic-acid-data ref]). S. cerevisae-genomet består av ca. 12,156*106 basepar. Basert på dette får vi da

200 *10-9 g DNA/ 12,156*106 bp) * (650 g/mol * bp) ~ 2,5*10-17 mol genomkopier.

Et mol er lik 6 * 1023. Vi har da

2,5 *10-17 mol * 6 * 1023 / mol ~ 1,5*107 (15 millioner) kopier.

S. Cerevisae har ca. 100-200 kopier av hvert rRNA-gen. Vi får da

15 millioner genomkopier * (~100 genkopier/genom) ~ 1,5 milliarder genkopier.

'''Extra reduced duration PCR program:'''

Tested 10.jul.2016. Amplification was successful for 1 of 2 samples.
Based on the results, further shortening to 25 cycles may be attempted.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 30 cycles

No final extension.

Total hold time: 63 min

Run time with OpenPCR: ~112 min. (1 h 52 minutes).

===Expected results===

'''Information from literature:'''

Expected PCR product size for ''S. cerevisiae'' (Strains CECT 1942 /ATCC 18824, CECT 1971) as reported by Esteve-Zarzoso et al: 880 bp.

'''Information from ''in silico'' PCR and published genomic sequence data:'''

[[Fil:ITS1 ITS4 S288C BLAST result.png|miniatyr|miniatyr|Graphic representation of BLAST result showing the location of the PCR target regions for the ITS1 and ITS4 primers in the S. cerevisae S288C genome (assembly R64, GenBank accession GCA_000146045.2). The query sequence is the expected PCR product sequence returned by the UCSC in silico PCR tool. While there are two target regions, there is only one expected product sequence as the sequence at and between the primer binding sites is identical for the two target regions. The locations of the target regions are shown as grey bands along the bottom, with the text "Query_185011" above each band. The left-pointing arrowing on each band indicates that the sequence of the query matches the sequence of the reverse strand as defined in the genome sequence file.]]

Expected fragment sizes as returned by in silico PCR at https://genome.ucsc.edu/cgi-bin/hgPcr (April 2011 assembly): 2 fragments of 841 bp each ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500810913_rmZAxNnexxkDaK66dHwLt86MeALA&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TCCGTAGGTGAACCTGCGG&wp_r=TCCTCCGCTTATTGATATGC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link]). The reported locations are chrXII:464259-465099 chrXII:455122-455962

The sequences of the two expected products as identified by in silico PCR above are identical. BLAST of the sequence against the R64 Assembly ([http://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/ GenBank GCA_000146045.2]) returns two matches, both on chromosome XII: Range 1: 455122 to 455962; and Range 2: 464259 to 465099. Both with 841/841 (100%) identities and zero gaps.

The genome assembly used for the in silico PCR described above is described as "Apr. 2011 (SacCer_Apr2011/sacCer3)". Although the relation or differences between this assembly/description and the R64 assembly have not been determined, the result of the BLAST search shows that the nucleotide positions on chromsome XII as reported in the ''in silico'' PCR result correspond to those of the R64 genome assembly and that there are no differences in the sequences of these regions. ([http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&QUERY=TCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTG%0AGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAG%0ATGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTG%0ATTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTT%0AGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTC%0AAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCT%0ACGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATC%0ATTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAAC%0AATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTT%0ATTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAAC%0ATGCGGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACA%0AATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATATCAATAAGCGGAGG%0AA&JOB_TITLE=Nucleotide%20Sequence%20%28841%20letters%29&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=Assembly&DATABASE=genomic/559292/GCF_000146045.2&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=4932&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=R&FILTER=m&WWW_BLAST_TYPE=newblast&EQ_MENU=Enter%20organism%20name%20or%20id--completions%20will%20be%20suggested&SHOW_OVERVIEW=true&SHOW_LINKOUT=true&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=true&NEW_VIEW=false&NCBI_GI=false&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&SHOW_CDS_FEATURE=false&OLD_BLAST=false direct link to search query])

(The sequence for S288C chromosome XII alone is accesible at: http://www.ncbi.nlm.nih.gov/nuccore/NC_001144.5)

[http://www.yeastgenome.org/browse/?loc=chrXII%3A463459..466018&tracks=DNA%2CAll%20Annotated%20Sequence%20Features&highlight=chrXII%3A464259..465099 Size & location of expected fragment 1, shown as highlighted area in SGD genome browser.]

[http://www.yeastgenome.org/browse/?loc=chrXII%3A454515..457224&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight=chrXII%3A455122..455962 Size & location of expected fragment 2, shown as highlighted area in SGD genome browser]

[[Fil:RDN582-region.png|700px|sentrer|Representation of the yeast genome region centered on the RDN58-2 rRNA gene, as shown in the SGD genome browser using the latest version of the S. cerevisae S288C genome (assembly R64). The S. cerevisae genome contains 100-200 repeats of the genes shown. The yellow region represents the target area for the ITS1-ITS4 primer combination in this location.]]

The target region spanning nucleotide positions 455122 to 455962 covers the 5.8S ribosomal RNA (rRNA) gene RDN58-1 (455414..455571) and the non-coding regions ITS2-1 and ITS1-1 which flank RDN58-1. Likewise, the target region spanning nucleotide positions 464259 to 465099 covers the 5.8S rRNA gene RDN58-2 (464551..464708) and the flanking non-coding regions ITS2-2 and ITS1-2.

SGD entry for RDN58-1: http://www.yeastgenome.org/locus/S000006488/overview

SGD entry for RDN58-2: http://www.yeastgenome.org/locus/S000006489/overview

RDN58-1 and RDN58-2 are contained within the [http://www.yeastgenome.org/locus/S000029411/overview RDN1 locus]. Although the sequence included in assembly R64 only indicates two target regions for the ITS1-ITS4 primer combination, the RDN1 locus in actuality represents a 1-2Mbp repeating region containing 100-200 rDNA repeats ([http://www.yeastgenome.org/locus/S000029411/overview ref]). Thus the actual number of targets can be expected to be in this range, and some sequence variation among these is likely ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665781/ ref]).

'''Expected PCR product sequence (841 bp):''' (NB: Whitespace is included below for easier viewing, adding extra characters)

Esteve-Zarzoso et al. used the restriction enzymes CfoI, HaeIII, and HinfI to cleave PCR product DNA and perform Restriction Fragment Length Polymorphism (RFLP) analysis. The sequence below contains 3 binding sites for CfoI (GCGC) CfoI, 3 binding sites for HaeIII (GGCC) and 3 binding sites for HinfI (GANTC, where N is any nucleotide). The sequence also contains an EcoRI binding site (GAATTC, bolded).

Esteve-Zarzoso reports the restriction fragment sizes for digestion with CfoI, HaeIII and HinfI as 385 + 365 (= 750), 320 + 230 + 180 + 150 (= 880) and 365 + 155 ( = 520), respectively (fragments smaller than 50 bp not included or reported).

Based on the sequence below, the expected fragment sizes from digestion are (http://www.restrictionmapper.org/):

*EcoRI 469 + 372 (= 841)
*CfoI: 363 + 334 + 134 + 10 (= 841)
*HaeIII: 311 + 230 + 172 + 128 (= 841)
*HinfI: 362 + 355 + 116 + 8 ( = 841)

<code>TCCGTAGGTGAACCTGCGGaaggatcattaaagaaatttaataattttga
aaatggatttttttgttttggcaagagcatgagagcttttactgggcaag
aagacaagagatggagagtccagccgggcctgcgcttaagtgcgcggtct
tgctaggcttgtaagtttctttcttgctattccaaacggtgagagatttc
tgtgcttttgttataggacaattaaaaccgtttcaatacaacacactgtg
gagttttcatatctttgcaactttttctttgggcattcgagcaatcgggg
cccagaggtaacaaacacaaacaattttatctattcattaaatttttgtc
aaaaacaagaattttcgtaactggaaattttaaaatattaaaaactttca
acaacggatctcttggttctcgcatcgatgaagaacgcagcgaaatgcga
tacgtaatgtgaattgca'''gaattc'''cgtgaatcatcgaatctttgaacgca
cattgcgccccttggtattccagggggcatgcctgtttgagcgtcatttc
cttctcaaacattctgtttggtagtgagtgatactctttggagttaactt
gaaattgctggccttttcattggatgttttttttccaaagagaggtttct
ctgcgtgcttgaggtataatgcaagtacggtcgttttaggttttaccaac
tgcggctaatctttttttatactgagcgtattggaacgttatcgataaga
agagagcgtctaggcgaacaatgttcttaaagtttgacctcaaatcaggt
aggagtacccgctgaacttaaGCATATCAATAAGCGGAGGA</code>

==V9D - LS266 yeast==

===Background===
"The primers V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used [to] amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA gene regions. These primers bind to conserved regions, with corresponding positions to Saccharomyces cerevisiae 18S (1609-1627) and 28S (287-266) rRNA genes." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

===PCR setup===

As described by Pryce (2010):

*Reaction volume: 50 uL
*Initial denaturation: 95 C for 9 min
*Repeated cycles:
*Denaturation: 95 c for 30s
*Anneal: 62C for 60s
*Extension: 72 C for 2 min
*Final extension: 72C for 5 min

x 33 (PCRS-B variant) or 35 (PCRS-D variant) cycles.

Total hold time: 136,5 min

===Expected results===
Expected fragment size: 1228 bp. ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500811567_RQun5ZzNd9dZSuQJcMHIctDo3PTE&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TTAAGTCCCTGCCCTTTGTA&wp_r=GCATTCCCAAACAACTCGACTC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link])

Expected fragment sizes after digestion by EcoRI: 628 + 600.

Expected PCR product sequence (S. cerevisiae):

<code>TTAcGTCCCTGCCCTTTGTAcacaccgcccgtcgctagtaccgattgaat
ggcttagtgaggcctcaggatctgcttagagaagggggcaactccatctc
agagcggagaatttggacaaacttggtcatttagaggaactaaaagtcgt
aacaaggtttccgtaggtgaacctgcggaaggatcattaaagaaatttaa
taattttgaaaatggatttttttgttttggcaagagcatgagagctttta
ctgggcaagaagacaagagatggagagtccagccgggcctgcgcttaagt
gcgcggtcttgctaggcttgtaagtttctttcttgctattccaaacggtg
agagatttctgtgcttttgttataggacaattaaaaccgtttcaatacaa
cacactgtggagttttcatatctttgcaactttttctttgggcattcgag
caatcggggcccagaggtaacaaacacaaacaattttatctattcattaa
atttttgtcaaaaacaagaattttcgtaactggaaattttaaaatattaa
aaactttcaacaacggatctcttggttctcgcatcgatgaagaacgcagc
gaaatgcgatacgtaatgtgaattgcagaattccgtgaatcatcgaatct
ttgaacgcacattgcgccccttggtattccagggggcatgcctgtttgag
cgtcatttccttctcaaacattctgtttggtagtgagtgatactctttgg
agttaacttgaaattgctggccttttcattggatgttttttttccaaaga
gaggtttctctgcgtgcttgaggtataatgcaagtacggtcgttttaggt
tttaccaactgcggctaatctttttttatactgagcgtattggaacgtta
tcgataagaagagagcgtctaggcgaacaatgttcttaaagtttgacctc
aaatcaggtaggagtacccgctgaacttaagcatatcaataagcggagga
aaagaaaccaaccgggattgccttagtaacggcgagtgaagcggcaaaag
ctcaaatttgaaatctggtaccttcggtgcccgagttgtaatttggagag
ggcaactttggggccgttccttgtctatgttccttggaacaggacgtcat
agagggtgagaatcccgtgtggcgaggagtgcggttctttgtaaagtgcc
ttcgaaGAGTCGAGTTGTTTGGGAATGC</code>

==MEATF + MEATR, animal meats==

===Background===

Lago et al. describe amplification and sequencing of the cytochrome b (cyt b) gene of several species using a set of degenerate primers. (European Food Research and Technology
March 2011, Volume 232, Issue 3, pp 509-515. http://link.springer.com/article/10.1007/s00217-010-1417-1)

===PCR setup===
Primers:
*MEAT F: CGAGGCCTMTAYTAYGG
*MEAT R: ATTGAKCGTAGGATTGCGTA

M denotes A or C. Y denotes C or T.

PCR program:

*Initial denaturation: 95C for 3 min
*Denaturation: 95 C for 30s
*Annealing: 50C for 30s
*Extension: 72 C for 30s

x 35 cycles.

*Final extension: 72C for 3 min

===Expected results===

Lago et al. report that DNA amplification with the primers MEAT F/R gave an amplicon of 555 bp in all tested species.

==PORCINE FWD + PORCINE REV, pork==

===Background===

See Ilhak and Arslan. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique. Turk. J. Vet. Anim. Sci. 2007; 31(3): 159-163: http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf

The porcine primers used by Ilhak and Arslan were designed based on Lahiff (2001). Mol Cell Probes. 2001 Feb;15(1):27-35. Species-specific PCR for the identification of ovine, porcine and chicken species in meta and bone meal (MBM).

See also GenBank accesion AF039170.1: http://www.ncbi.nlm.nih.gov/nuccore/AF039170

===PCR setup===

Primers:
*Porcine FWD: GCC TAA ATC TCC CCT CAA TGG TA
*Porcine REV: ATGAAAGAGGCAAATAGATTTTCG

*Reaction volume: 50 uL
*20 pmol of each primer

PCR program:
*Denaturation: 94 C 45 s
*Annealing: 58C 45 s
*Extension: 72C 90 s.

x 30 cycles.

===Electrophoresis===

15 uL PCR product, 1.5 % agarose at 100 V for 2 h.

===Expected results===

Ilhak and Arslan report a PCR product size of 212 bp for porcine meat.

=Lagring av bakterie-stammer i glycerol=

*1 Prepare a solution of 60 % v/v glycerol in water. (For 25 mL, mix 10 mL water and 15 mL glycerol)
*2 Add 400 uL 60 % glycerol solution and 800 uL of the culture to be stored in a cryogenic tube.
*3 Mix thoroughly!
*4 Place in 5 C refrigerator for 30 min, then move to -80 C freezer.

=Sekvenseringstjenester=

==GATC (lightrun)==
Primer specifications:
*Tm 52-58 C
*17-19 bp
*G or C at 3' end (max 3 Gs or Cs)
*maximum 4 identical sequential bp.

Preparation instructions:
*Add 5 uL template DNA (80-100 ng/uL plasmid DNA or 20-80 ng/uL purified PCR product) and 5 uL primer, 5 uM (5 pmol/uL) in one 1.5 mL tube.
*Drop off at GATC collection point or ship to: GATC Biotech AG. European Custom Sequencing Centre. Gotrfied-Hagen-Strasse 20. 51105 Köln.

==Macrogen==

Primer specifications:
*18-25 bp
*40-60 % GC
*Tm 55-60

Preparation instructions:
*Add 20 uL DNA (100 ng/uL plasmid or 50 ng/uL purified PCR product) to one tube. Add 20µl primer (10 pmol/uL) to a separate tube (?)
*Ship to: Macrogen Europe, IWO, Kamer IA3-195, Meibergdreef 39,1105 AZ Amsterdam Zuid-oost. Netherlands. Attention: J.S .Park

Preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

=Brukermanualer=

BioRad PowerPac Basic Power Supply: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006213.pdf

BioHackerLab/Protokoller

2016-08-07T23:12:11Z

Jarlemag: /* Background */

=Resuspendering av DNA-primere=

Ta røret med tørket DNA ut av pakken. Kontroller at ID og DNA-sekvens oppgitt på røret stemmer med bestillingen.

Plasser røret i en mikrosentrifuge og sentrifuger i ca. 30 sekunder.

Tilsett NF-vann til konsentrasjon etter oppløsning lik 100 pmol/uL (0.1 mM, 100 uM). Se siden av røret eller medfølgende dokumentasjon fra leverandør for total mengde DNA i røret og/eller mengde vann som må tilsettes for endelig konsentrasjon ~100 pmol/uL

Bland ved å knipse på og riste flasken, eller med en vortex-mikser. Sentrifuger til slutt røret igjen.

Oppbevar fryst, fortrinnsvis ved -20C.

Før bruk til PCR vil det være hensiktsmessig å foreta ytterligere 10X fortynning til 10 pmol/uL (10 uM) i et eget rør, som også kan oppbevares fryst og brukes flere ganger. Dette reduserer også behovet for å åpne hovedrøret og reduserer risikoen for kontaminasjon av dette.

Se også https://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html?

Om mulig kontroller konsentrasjonen vha. spektrofotometri. Se http://www.promega.com/a/apps/biomath/index.html?calc=odConvert

=DNA extraction=

Blount, Driessen & Ellis: GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA. Scientific Reports 6, Article number: 26863 (2016). http://www.nature.com/articles/srep26863

Strawberry crude DNA extraction: http://www.stevespanglerscience.com/lab/experiments/strawberry-dna/

=DNA quantification=

==Background==

The main methods for determiniing nucleic acid concentrations are UV photometry measuring absorbance at 260 nm (A260) and fluorometry. Note that different sources may describe different conversion factors for conversion from A260 to mass concentration. For oligomers, a more accurate DNA concentration value may be achieved by estimating the sequence-dependent extinction coefficient by calculation.

http://biotools.nubic.northwestern.edu/OligoCalc.html

http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full

http://www.promega.com/a/apps/biomath/

https://no.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/

http://www.ogt.co.uk/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality

https://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/09%20UV%20Absorption.pdf

https://www.idtdna.com/pages/support/technical-vault/reading-room/quick-reference/quick-reference/2011/06/02/molar-extinction-coefficient

http://www.endmemo.com/bio/OD260.php

=Elektroforese=

Se også: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_SeaKem_LE_Agarose_-_Protocol.pdf

'''OBS: Les gjennom hele protokollen før gjennomførelse for å bli kjent med relevant sikkerhetsinformasjon.'''

==Utstyr==

'''OBS: Kontroller utstyret for skader og slitasje før bruk. Ikke fortsett dersom elektroforesekammer, ledninger eller strømforsyning viser tegn til skade.'''

*Elektroforesekammer. Eks. Carolina Deluxe Gel Chamber.
*Strømforsyning. Eks. BioRad PowerPac Basic
*Automatpipette, 1-10 uL.
*Vekt, 0.1 g eller høyere oppløsning.
*Mikrobølgeovn
*Varmebeskyttende hansker eller annen håndbeskyttelse
*Målesylinder, 50 mL eller større
*Glassflaske til agaroseløsning, 100 mL eller større (må passe i mikrobølgeovn)
*Glassflaske til TAE-bufferløsning, 250 mL eller større
*Transilluminator, eks. DarkReader blue light.
*Stekespade eller lignende for å løfte agarosegel

Avfallsbeholder for pipettespisser og tørt avfall
Avfallsbeholder for brukt buffer/flytende avfall

Forbruksvarer:
*Agarose for elektroforese, eks. SeaKem LE agarose. Ca. 0.5 g/gel.
*TAE 10x buffer. Ca. 30 mL.
*Destillert vann: Ca. 300 mL.
*DNA-fargestoff, eks. GelGreen. Ca 5 uL.
*Pipettespisser, 1-10 uL.
*Veieskip.
*Tørkepapir
*DNA ladder
*Loading dye

==Støping av agarose-gel==

Om nødvendig, lag TAE buffer i brukskonsentrasjon ved å fortynne konsentrert buffer i destillert vann. For å lage 1x TAE buffer fra 10x TAE buffer, bland 1 del TAE buffer og 9 deler destillert vann. TAE buffer er irriterende. Bruk hansker ved håndtering av konsentrert buffer.

Tilsett gradvis ~1 v/v % agarose til 1x TAE buffer (~1g til 100 mL, f.eks 0.5g for 50 mL) i en flaske med volum minst to ganger volumet av løsningen, fortrinnsvis under omrøring med magnetrører - eventuelt rør for hånd med glasstav eller ved å bevege flasken.

'''OBS: Flasken må ikke være lufttett!'''

Varm løsningen i mikrobølgeovn på høy styrke til løsningen koker (ca. ett minutt). La løsningen koke i ca. 30 sekunder. Reduser varmeeffekten eller skru av ovnen dersom løsningen koker over.

Fjern flasken fra mikrobølgeovnen. Bland løsningen ved å bevege flasken '''forsiktig''' i en roterende bevegelse for hånd, eller med en magnetrører. Utvis forsiktighet for å unngå sprut. Kontroller at agarose-løsningen er klar og uten synlige partikler. Dersom det er partikler og uløst agarose i løsningen, kok løsningen igjen.

'''OBS: Bruk øyebeskyttelse! Fare for støtkoking og sprut også etter at oppvarmingen har opphørt. Flasken er varm. Bruk varmebeskyttende hansker eller annen håndbeskyttelse ved håndtering av flaske. Bruk fortrinnsvis labfrakk eller langarmede klær og unngå eksponering av bar hud. Fare for forbrenning ved søl eller sprut.'''

Tilsett DNA-fargestoff i henhold til konsentrasjonsangivelse. F.eks, for 10 000x konsentrert fargestoff, tilsett 1 uL fargestoff per 10 mL agarose løsning (5 uL for 50 mL).

Plasser et gel-støpekar på tvers av lengderetningen i et elektroforesekar og plasser en brønnkam i støpekaret. Hell varm agaroseløsning i kammeret og la stå til gel'en er størknet (ca. 30-60 minutter).

'''OBS: Elektroforesekaret skal være frakoblet fra strømforsyning når gel'en støpes.'''

Ta ut brønnkammen og snu støpekaret slik at prøvebrønnene er nærmest den sorte (postive) elektroden.

Hell 1x TAE buffer i elektroforesekaret slik at gel'en er dekket av buffer.

==Elektroforese==

*Tilsett prøvene i prøvebrønnene.
*Sett på lokket på elektroforesekaret.
*Kontroller at området rundt elektroforesekaret og strømforsyningen er tørt. Tørk bort eventuell væske.
*Koble ledningene til strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.

'''OBS: Strømforsyningen skal være avslått når ledningene kobles til!'''

*Skru på strømforsyningen.
*Juster til ønsket spenning og skru på spenningen.
*La elektroforesen foregå uforstyrret. '''Ikke rør kammeret eller ledningene så lenge spenningen er på. Dersom det oppstår tegn til lekkasje fra kammeret, avslutt elektroforesen umiddelbart uten å komme i kontakt med væsken.'''
*Skru av spenningen på strømforsyningen.
*Skru av strømforsyningen.
*Koble ledningene fra strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.
*Ta av lokket på elektroforesekaret.
*FLytt agarosegeleen forsiktig til en transilluminator ved hjelp av en stekespade e.l.
*Avhend agarose-gel, bufferløsning og forbruksmateriell på forsvarlig vis etter bruk. Skyll elektroforesekammer og glassutstyr med destillert vann.

Se også: https://www.addgene.org/plasmid-protocols/gel-electrophoresis/

http://www.methodbook.net/dna/agarogel.html

Typisk prøvevolum kan være 6 uL, f.eks 5 uL PCR-produkt + 1 uL 6x loading dye, eller 4 uL vann, 1 uL DNA ladder og 1 uL 6x loading dye.

=PCR=

Typiske reaksjonsvolum er 20 uL og 50 uL. Gitt en konsentrasjon i primerløsningen lik 10 uM vil i de to tilfellene tilsetting av henholdsvis 1 uL og 2.5 uL av hver primer gi en endelig kosentrasjon av hver primer lik 0.5 uM i reaksjonsblandingen.

http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Typisk vil en ta ut ca. 5 uL av reaksjonsblandingen til elektroforese-analyse etter PCR.

Se også https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

==Detection of D1S80 Repeat Polymorphism by PCR==

See https://www.dnalc.org/files/pdf/forensicprofchip_d1s80_protocol.pdf

==Collection of genomic DNA by buccal swabs==

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566681/pdf/envhper00512-0045.pdf

==PTC tasting ability genotyping==

https://www.snpedia.com/index.php/Rs713598

https://www.ncbi.nlm.nih.gov/pubmed/12595690?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181941/

http://www.ncbi.nlm.nih.gov/gene?cmd=retrieve&dopt=default&rn=1&list_uids=5726

http://bioinformatics.dnalc.org/ptc/animation/pdf/ptc.pdf

==Sex determination by PCR==

See https://en.wikipedia.org/wiki/Amelogenin#Application_in_sex_determination and Fracnes et al.: Clin Chim Acta. 2007 Nov-Dec;386(1-2):53-6. Epub 2007 Jul 31.
Amelogenin test: From forensics to quality control in clinical and biochemical genomics.. (http://www.sciencedirect.com/science/article/pii/S0009898107003889)

Primers:

*AME-F: 5'-ctgatggttggcctcaagcctgtg-3'
*AME-R: 5'-taaagagattcattaacttgactg-3'

Expected results:

*If male: 1 band of 977 bp + 1 band of 790 bp
*If female: 1 band of 977 bp

==DNA fingerprinting==

===CODIS===

The Combined DNA Index System (CODIS) is a FBI program to support law enforcement by DNA-based identification. CODIS defines standards for DNA fingerprinting and a selection of genetic loci to be analyzed as part of DNA fingerprinting. The 13 CODIS core loci per 2016 are:

*CSF1PO
*FGA
*THO1
*TPOX
*VWA
*D3S1358
*D5S818
*D7S820
*D8S1179
*D13S317
*D16S539
*D18S51
*D21S11

In addition, the Amelogenin (AMEL) locus is used for sex determination.

For information on each locus, see http://www.cstl.nist.gov/strbase/

See also http://www.cstl.nist.gov/strbase/coreSTRs.htm

7 additional loci will be required from January 1 2017.

http://isogg.org/wiki/CODIS

https://en.wikipedia.org/wiki/Combined_DNA_Index_System

http://www.cstl.nist.gov/biotech/strbase/fbicore.htm

http://www.cybertory.org/resources/CODIS/

Estimated cost for CODIS primer set as described by cybertory.org, with synthesis by Macrogen Inc.: 68.2 EUR / ~75 USD / ~ 650 NOK + shipping.

===European set===

The European Standard Set (ESS) consists of the following STR loci: FGA, TH01, VWA, D1S1656, D2S441, D3S1358, D8S1179, D10S1248, D12S391, D18S51, D21S11, D22S1045

Additional Loci commonly found in European STR kits include: D2S1338, D16S539, D19S433, SE33, Amelogenin

==Colony PCR==

1. From an agar plate, select one or several colonies for colony PCR. For each colony selected, pick a small amount of colony material using a sterile pipette tip, and dissolve in 50 uL H2O. Reseed a new agar plate with 5 uL of the resulting solution(s) each in a separate spot, keeping the spots separate and noting the location of each. Alternatively, use 5 uL to inoculate a liquid culture.

Incubate the dissolved colony material at 96 C for 10 min to release DNA.

Use 1 uL of the heat-treated solution as template for PCR.

==ITS1 + ITS 4 yeast==

===Background===

See Esteve-Zarzoso et al.: ''Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers''. (http://www.ncbi.nlm.nih.gov/pubmed/10028278)

[https://www.researchgate.net/publication/13261852_Esteve-Zarzoso_B_Belloch_C_Uruburu_F_Querol_A_Identification_of_yeasts_by_RFLP_analysis_of_the_58S_rRNA_gene_and_the_two_ribosomal_internal_transcribed_spacers_Int_J_Syst_Bacteriol_49_329-337 Artikkel tilgjengelig via ResearchGate]

See also http://sites.biology.duke.edu/fungi/mycolab/primers.htm

[[Fil:ITS1 ITS4 insilico UCSC.png|miniatyr|høyre|Screenshot of in silico PCR result using the UCSC in silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr) with the primer sequences ITS1 (5' TCCGTAGGTGAACCTGCGG 3') and ITS4 (5' TCCTCCGCTTATTGATATGC 3'), selecting the S. cerevisae April 2011 (SacCer_Apr2011/sacCer3) genome assembly as the template. The result shows two expected PCR products/target regions with identical sequences. For each, the line starting with a > specifies the target location in the assembly, the target region size/expected product size, and the primer sequences, separated by spaces. The subsequent lines show the target region/expected product sequence, with the sequence of the forward primer (ITS1) and the sequence matching the reverse primer (ITS4) in capitals. The target region size/expected product size (841bp) includes the primers.]]

===PCR setup===

'''According to Esteve-Zarzoso et al.:'''

Reaction volume: 100 uL

Primer concentrations: 0.5 uM each

Forward (FWD) primer (ITS1): 5' TCCGTAGGTGAACCTGCGG 3'

Reverse (REV) primer (ITS4): 5' TCCTCCGCTTATTGATATGC 3'

(Start and end of expected product sequence = FWD primer + reverse complement of reverse primer: TCCGTAGGTGAACCTGCGG-GCATATCAATAAGCGGAGGA)

Template: Fresh yeast colony material.

Template preparation: 95 C for 15 min.

Initial denaturation: 95C for 5 min

Repeated cycles:
*Denaturation: 94 C for 1 min
*Anneal: 55.5 C for 2 min
*Extension 72C for 2 min

x 35 cycles

Final extension: 72C for 10 min

Total hold time: 190 min

Estimated program duration with OpenPCR: ~4 hr.

'''Alternative setup and reduced duration PCR program:'''

Reaction volume: 50 uL

Template: The protocol has been tested with dry yeast, see template preparation below. The protocol may also be attempted with fresh store-bought yeast, liquid yeast culture or yeast agar colony material as the template source.

PCR setup otherwise as original protocol.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 35 cycles

Step durations and temperatures are according to recommendations for Dongsheng Taq polymerase. Annealing temperature according to original protocol.

Final extension: 72 C for 10 min

Total hold time: 83 min

Run time with OpenPCR: ~ 2h 20 min.

'''Template preparation:'''

Dissolve 0.1g dry yeast (Idun tørrgjær) in 10 mL dH20. Mix well. Pipette 50 uL (-> ~0.0005g, 0.5mg yeast powder by dry weight) into a microsentrifuge tube or PCR tube and incubate at 98C for 10 minutes. Use 1-2 uL (-> ~0,00002g = 0,02mg = 20 ug yeast powder by dry weight for 2 uL) of the supernatant as template.

I tørrvekt kan det forventes at DNA utgjør i størrelsesorden 1% av cellemassen. ([http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/ ref])
20 ug tørrmasse vil da gi anslagsvis ~0.2 ug = 200 ng DNA.

DNA har molekylær vekt lik ca. 650 g/mol per basepar. ([https://www.neb.com/tools-and-resources/usage-guidelines/nucleic-acid-data ref]). S. cerevisae-genomet består av ca. 12,156*106 basepar. Basert på dette får vi da

200 *10-9 g DNA/ 12,156*106 bp) * (650 g/mol * bp) ~ 2,5*10-17 mol genomkopier.

Et mol er lik 6 * 1023. Vi har da

2,5 *10-17 mol * 6 * 1023 / mol ~ 1,5*107 (15 millioner) kopier.

S. Cerevisae har ca. 100-200 kopier av hvert rRNA-gen. Vi får da

15 millioner genomkopier * (~100 genkopier/genom) ~ 1,5 milliarder genkopier.

'''Extra reduced duration PCR program:'''

Tested 10.jul.2016. Amplification was successful for 1 of 2 samples.
Based on the results, further shortening to 25 cycles may be attempted.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 30 cycles

No final extension.

Total hold time: 63 min

Run time with OpenPCR: ~112 min. (1 h 52 minutes).

===Expected results===

'''Information from literature:'''

Expected PCR product size for ''S. cerevisiae'' (Strains CECT 1942 /ATCC 18824, CECT 1971) as reported by Esteve-Zarzoso et al: 880 bp.

'''Information from ''in silico'' PCR and published genomic sequence data:'''

[[Fil:ITS1 ITS4 S288C BLAST result.png|miniatyr|miniatyr|Graphic representation of BLAST result showing the location of the PCR target regions for the ITS1 and ITS4 primers in the S. cerevisae S288C genome (assembly R64, GenBank accession GCA_000146045.2). The query sequence is the expected PCR product sequence returned by the UCSC in silico PCR tool. While there are two target regions, there is only one expected product sequence as the sequence at and between the primer binding sites is identical for the two target regions. The locations of the target regions are shown as grey bands along the bottom, with the text "Query_185011" above each band. The left-pointing arrowing on each band indicates that the sequence of the query matches the sequence of the reverse strand as defined in the genome sequence file.]]

Expected fragment sizes as returned by in silico PCR at https://genome.ucsc.edu/cgi-bin/hgPcr (April 2011 assembly): 2 fragments of 841 bp each ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500810913_rmZAxNnexxkDaK66dHwLt86MeALA&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TCCGTAGGTGAACCTGCGG&wp_r=TCCTCCGCTTATTGATATGC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link]). The reported locations are chrXII:464259-465099 chrXII:455122-455962

The sequences of the two expected products as identified by in silico PCR above are identical. BLAST of the sequence against the R64 Assembly ([http://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/ GenBank GCA_000146045.2]) returns two matches, both on chromosome XII: Range 1: 455122 to 455962; and Range 2: 464259 to 465099. Both with 841/841 (100%) identities and zero gaps.

The genome assembly used for the in silico PCR described above is described as "Apr. 2011 (SacCer_Apr2011/sacCer3)". Although the relation or differences between this assembly/description and the R64 assembly have not been determined, the result of the BLAST search shows that the nucleotide positions on chromsome XII as reported in the ''in silico'' PCR result correspond to those of the R64 genome assembly and that there are no differences in the sequences of these regions. ([http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&QUERY=TCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTG%0AGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAG%0ATGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTG%0ATTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTT%0AGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTC%0AAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCT%0ACGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATC%0ATTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAAC%0AATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTT%0ATTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAAC%0ATGCGGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACA%0AATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATATCAATAAGCGGAGG%0AA&JOB_TITLE=Nucleotide%20Sequence%20%28841%20letters%29&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=Assembly&DATABASE=genomic/559292/GCF_000146045.2&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=4932&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=R&FILTER=m&WWW_BLAST_TYPE=newblast&EQ_MENU=Enter%20organism%20name%20or%20id--completions%20will%20be%20suggested&SHOW_OVERVIEW=true&SHOW_LINKOUT=true&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=true&NEW_VIEW=false&NCBI_GI=false&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&SHOW_CDS_FEATURE=false&OLD_BLAST=false direct link to search query])

(The sequence for S288C chromosome XII alone is accesible at: http://www.ncbi.nlm.nih.gov/nuccore/NC_001144.5)

[http://www.yeastgenome.org/browse/?loc=chrXII%3A463459..466018&tracks=DNA%2CAll%20Annotated%20Sequence%20Features&highlight=chrXII%3A464259..465099 Size & location of expected fragment 1, shown as highlighted area in SGD genome browser.]

[http://www.yeastgenome.org/browse/?loc=chrXII%3A454515..457224&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight=chrXII%3A455122..455962 Size & location of expected fragment 2, shown as highlighted area in SGD genome browser]

[[Fil:RDN582-region.png|700px|sentrer|Representation of the yeast genome region centered on the RDN58-2 rRNA gene, as shown in the SGD genome browser using the latest version of the S. cerevisae S288C genome (assembly R64). The S. cerevisae genome contains 100-200 repeats of the genes shown. The yellow region represents the target area for the ITS1-ITS4 primer combination in this location.]]

The target region spanning nucleotide positions 455122 to 455962 covers the 5.8S ribosomal RNA (rRNA) gene RDN58-1 (455414..455571) and the non-coding regions ITS2-1 and ITS1-1 which flank RDN58-1. Likewise, the target region spanning nucleotide positions 464259 to 465099 covers the 5.8S rRNA gene RDN58-2 (464551..464708) and the flanking non-coding regions ITS2-2 and ITS1-2.

SGD entry for RDN58-1: http://www.yeastgenome.org/locus/S000006488/overview

SGD entry for RDN58-2: http://www.yeastgenome.org/locus/S000006489/overview

RDN58-1 and RDN58-2 are contained within the [http://www.yeastgenome.org/locus/S000029411/overview RDN1 locus]. Although the sequence included in assembly R64 only indicates two target regions for the ITS1-ITS4 primer combination, the RDN1 locus in actuality represents a 1-2Mbp repeating region containing 100-200 rDNA repeats ([http://www.yeastgenome.org/locus/S000029411/overview ref]). Thus the actual number of targets can be expected to be in this range, and some sequence variation among these is likely ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665781/ ref]).

'''Expected PCR product sequence (841 bp):''' (NB: Whitespace is included below for easier viewing, adding extra characters)

Esteve-Zarzoso et al. used the restriction enzymes CfoI, HaeIII, and HinfI to cleave PCR product DNA and perform Restriction Fragment Length Polymorphism (RFLP) analysis. The sequence below contains 3 binding sites for CfoI (GCGC) CfoI, 3 binding sites for HaeIII (GGCC) and 3 binding sites for HinfI (GANTC, where N is any nucleotide). The sequence also contains an EcoRI binding site (GAATTC, bolded).

Esteve-Zarzoso reports the restriction fragment sizes for digestion with CfoI, HaeIII and HinfI as 385 + 365 (= 750), 320 + 230 + 180 + 150 (= 880) and 365 + 155 ( = 520), respectively (fragments smaller than 50 bp not included or reported).

Based on the sequence below, the expected fragment sizes from digestion are (http://www.restrictionmapper.org/):

*EcoRI 469 + 372 (= 841)
*CfoI: 363 + 334 + 134 + 10 (= 841)
*HaeIII: 311 + 230 + 172 + 128 (= 841)
*HinfI: 362 + 355 + 116 + 8 ( = 841)

<code>TCCGTAGGTGAACCTGCGGaaggatcattaaagaaatttaataattttga
aaatggatttttttgttttggcaagagcatgagagcttttactgggcaag
aagacaagagatggagagtccagccgggcctgcgcttaagtgcgcggtct
tgctaggcttgtaagtttctttcttgctattccaaacggtgagagatttc
tgtgcttttgttataggacaattaaaaccgtttcaatacaacacactgtg
gagttttcatatctttgcaactttttctttgggcattcgagcaatcgggg
cccagaggtaacaaacacaaacaattttatctattcattaaatttttgtc
aaaaacaagaattttcgtaactggaaattttaaaatattaaaaactttca
acaacggatctcttggttctcgcatcgatgaagaacgcagcgaaatgcga
tacgtaatgtgaattgca'''gaattc'''cgtgaatcatcgaatctttgaacgca
cattgcgccccttggtattccagggggcatgcctgtttgagcgtcatttc
cttctcaaacattctgtttggtagtgagtgatactctttggagttaactt
gaaattgctggccttttcattggatgttttttttccaaagagaggtttct
ctgcgtgcttgaggtataatgcaagtacggtcgttttaggttttaccaac
tgcggctaatctttttttatactgagcgtattggaacgttatcgataaga
agagagcgtctaggcgaacaatgttcttaaagtttgacctcaaatcaggt
aggagtacccgctgaacttaaGCATATCAATAAGCGGAGGA</code>

==V9D - LS266 yeast==

===Background===
"The primers V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used [to] amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA gene regions. These primers bind to conserved regions, with corresponding positions to Saccharomyces cerevisiae 18S (1609-1627) and 28S (287-266) rRNA genes." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

===PCR setup===

As described by Pryce (2010):

*Reaction volume: 50 uL
*Initial denaturation: 95 C for 9 min
*Repeated cycles:
*Denaturation: 95 c for 30s
*Anneal: 62C for 60s
*Extension: 72 C for 2 min
*Final extension: 72C for 5 min

x 33 (PCRS-B variant) or 35 (PCRS-D variant) cycles.

Total hold time: 136,5 min

===Expected results===
Expected fragment size: 1228 bp. ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500811567_RQun5ZzNd9dZSuQJcMHIctDo3PTE&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TTAAGTCCCTGCCCTTTGTA&wp_r=GCATTCCCAAACAACTCGACTC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link])

Expected fragment sizes after digestion by EcoRI: 628 + 600.

Expected PCR product sequence (S. cerevisiae):

<code>TTAcGTCCCTGCCCTTTGTAcacaccgcccgtcgctagtaccgattgaat
ggcttagtgaggcctcaggatctgcttagagaagggggcaactccatctc
agagcggagaatttggacaaacttggtcatttagaggaactaaaagtcgt
aacaaggtttccgtaggtgaacctgcggaaggatcattaaagaaatttaa
taattttgaaaatggatttttttgttttggcaagagcatgagagctttta
ctgggcaagaagacaagagatggagagtccagccgggcctgcgcttaagt
gcgcggtcttgctaggcttgtaagtttctttcttgctattccaaacggtg
agagatttctgtgcttttgttataggacaattaaaaccgtttcaatacaa
cacactgtggagttttcatatctttgcaactttttctttgggcattcgag
caatcggggcccagaggtaacaaacacaaacaattttatctattcattaa
atttttgtcaaaaacaagaattttcgtaactggaaattttaaaatattaa
aaactttcaacaacggatctcttggttctcgcatcgatgaagaacgcagc
gaaatgcgatacgtaatgtgaattgcagaattccgtgaatcatcgaatct
ttgaacgcacattgcgccccttggtattccagggggcatgcctgtttgag
cgtcatttccttctcaaacattctgtttggtagtgagtgatactctttgg
agttaacttgaaattgctggccttttcattggatgttttttttccaaaga
gaggtttctctgcgtgcttgaggtataatgcaagtacggtcgttttaggt
tttaccaactgcggctaatctttttttatactgagcgtattggaacgtta
tcgataagaagagagcgtctaggcgaacaatgttcttaaagtttgacctc
aaatcaggtaggagtacccgctgaacttaagcatatcaataagcggagga
aaagaaaccaaccgggattgccttagtaacggcgagtgaagcggcaaaag
ctcaaatttgaaatctggtaccttcggtgcccgagttgtaatttggagag
ggcaactttggggccgttccttgtctatgttccttggaacaggacgtcat
agagggtgagaatcccgtgtggcgaggagtgcggttctttgtaaagtgcc
ttcgaaGAGTCGAGTTGTTTGGGAATGC</code>

==MEATF + MEATR, animal meats==

===Background===

Lago et al. describe amplification and sequencing of the cytochrome b (cyt b) gene of several species using a set of degenerate primers. (European Food Research and Technology
March 2011, Volume 232, Issue 3, pp 509-515. http://link.springer.com/article/10.1007/s00217-010-1417-1)

===PCR setup===
Primers:
*MEAT F: CGAGGCCTMTAYTAYGG
*MEAT R: ATTGAKCGTAGGATTGCGTA

M denotes A or C. Y denotes C or T.

PCR program:

*Initial denaturation: 95C for 3 min
*Denaturation: 95 C for 30s
*Annealing: 50C for 30s
*Extension: 72 C for 30s

x 35 cycles.

*Final extension: 72C for 3 min

===Expected results===

Lago et al. report that DNA amplification with the primers MEAT F/R gave an amplicon of 555 bp in all tested species.

==PORCINE FWD + PORCINE REV, pork==

===Background===

See Ilhak and Arslan. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique. Turk. J. Vet. Anim. Sci. 2007; 31(3): 159-163: http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf

The porcine primers used by Ilhak and Arslan were designed based on Lahiff (2001). Mol Cell Probes. 2001 Feb;15(1):27-35. Species-specific PCR for the identification of ovine, porcine and chicken species in meta and bone meal (MBM).

See also GenBank accesion AF039170.1: http://www.ncbi.nlm.nih.gov/nuccore/AF039170

===PCR setup===

Primers:
*Porcine FWD: GCC TAA ATC TCC CCT CAA TGG TA
*Porcine REV: ATGAAAGAGGCAAATAGATTTTCG

*Reaction volume: 50 uL
*20 pmol of each primer

PCR program:
*Denaturation: 94 C 45 s
*Annealing: 58C 45 s
*Extension: 72C 90 s.

x 30 cycles.

===Electrophoresis===

15 uL PCR product, 1.5 % agarose at 100 V for 2 h.

===Expected results===

Ilhak and Arslan report a PCR product size of 212 bp for porcine meat.

=Lagring av bakterie-stammer i glycerol=

*1 Prepare a solution of 60 % v/v glycerol in water. (For 25 mL, mix 10 mL water and 15 mL glycerol)
*2 Add 400 uL 60 % glycerol solution and 800 uL of the culture to be stored in a cryogenic tube.
*3 Mix thoroughly!
*4 Place in 5 C refrigerator for 30 min, then move to -80 C freezer.

=Sekvenseringstjenester=

==GATC (lightrun)==
Primer specifications:
*Tm 52-58 C
*17-19 bp
*G or C at 3' end (max 3 Gs or Cs)
*maximum 4 identical sequential bp.

Preparation instructions:
*Add 5 uL template DNA (80-100 ng/uL plasmid DNA or 20-80 ng/uL purified PCR product) and 5 uL primer, 5 uM (5 pmol/uL) in one 1.5 mL tube.
*Drop off at GATC collection point or ship to: GATC Biotech AG. European Custom Sequencing Centre. Gotrfied-Hagen-Strasse 20. 51105 Köln.

==Macrogen==

Primer specifications:
*18-25 bp
*40-60 % GC
*Tm 55-60

Preparation instructions:
*Add 20 uL DNA (100 ng/uL plasmid or 50 ng/uL purified PCR product) to one tube. Add 20µl primer (10 pmol/uL) to a separate tube (?)
*Ship to: Macrogen Europe, IWO, Kamer IA3-195, Meibergdreef 39,1105 AZ Amsterdam Zuid-oost. Netherlands. Attention: J.S .Park

Preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

=Brukermanualer=

BioRad PowerPac Basic Power Supply: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006213.pdf

BioHackerLab/Protokoller

2016-08-07T23:00:14Z

Jarlemag:

=Resuspendering av DNA-primere=

Ta røret med tørket DNA ut av pakken. Kontroller at ID og DNA-sekvens oppgitt på røret stemmer med bestillingen.

Plasser røret i en mikrosentrifuge og sentrifuger i ca. 30 sekunder.

Tilsett NF-vann til konsentrasjon etter oppløsning lik 100 pmol/uL (0.1 mM, 100 uM). Se siden av røret eller medfølgende dokumentasjon fra leverandør for total mengde DNA i røret og/eller mengde vann som må tilsettes for endelig konsentrasjon ~100 pmol/uL

Bland ved å knipse på og riste flasken, eller med en vortex-mikser. Sentrifuger til slutt røret igjen.

Oppbevar fryst, fortrinnsvis ved -20C.

Før bruk til PCR vil det være hensiktsmessig å foreta ytterligere 10X fortynning til 10 pmol/uL (10 uM) i et eget rør, som også kan oppbevares fryst og brukes flere ganger. Dette reduserer også behovet for å åpne hovedrøret og reduserer risikoen for kontaminasjon av dette.

Se også https://www.thermofisher.com/no/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html?

Om mulig kontroller konsentrasjonen vha. spektrofotometri. Se http://www.promega.com/a/apps/biomath/index.html?calc=odConvert

=DNA extraction=

Blount, Driessen & Ellis: GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA. Scientific Reports 6, Article number: 26863 (2016). http://www.nature.com/articles/srep26863

Strawberry crude DNA extraction: http://www.stevespanglerscience.com/lab/experiments/strawberry-dna/

=DNA quantification=

==Background==

The main methods for determiniing nucleic acid concentrations are UV photometry measuring absorbance at 260 nm (A260) and fluorometry. Note that different sources may describe different conversion factors for conversion from A260 to mass concentration.

http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full

http://www.promega.com/a/apps/biomath/

https://no.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/

http://www.ogt.co.uk/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality

https://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/09%20UV%20Absorption.pdf

=Elektroforese=

Se også: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_SeaKem_LE_Agarose_-_Protocol.pdf

'''OBS: Les gjennom hele protokollen før gjennomførelse for å bli kjent med relevant sikkerhetsinformasjon.'''

==Utstyr==

'''OBS: Kontroller utstyret for skader og slitasje før bruk. Ikke fortsett dersom elektroforesekammer, ledninger eller strømforsyning viser tegn til skade.'''

*Elektroforesekammer. Eks. Carolina Deluxe Gel Chamber.
*Strømforsyning. Eks. BioRad PowerPac Basic
*Automatpipette, 1-10 uL.
*Vekt, 0.1 g eller høyere oppløsning.
*Mikrobølgeovn
*Varmebeskyttende hansker eller annen håndbeskyttelse
*Målesylinder, 50 mL eller større
*Glassflaske til agaroseløsning, 100 mL eller større (må passe i mikrobølgeovn)
*Glassflaske til TAE-bufferløsning, 250 mL eller større
*Transilluminator, eks. DarkReader blue light.
*Stekespade eller lignende for å løfte agarosegel

Avfallsbeholder for pipettespisser og tørt avfall
Avfallsbeholder for brukt buffer/flytende avfall

Forbruksvarer:
*Agarose for elektroforese, eks. SeaKem LE agarose. Ca. 0.5 g/gel.
*TAE 10x buffer. Ca. 30 mL.
*Destillert vann: Ca. 300 mL.
*DNA-fargestoff, eks. GelGreen. Ca 5 uL.
*Pipettespisser, 1-10 uL.
*Veieskip.
*Tørkepapir
*DNA ladder
*Loading dye

==Støping av agarose-gel==

Om nødvendig, lag TAE buffer i brukskonsentrasjon ved å fortynne konsentrert buffer i destillert vann. For å lage 1x TAE buffer fra 10x TAE buffer, bland 1 del TAE buffer og 9 deler destillert vann. TAE buffer er irriterende. Bruk hansker ved håndtering av konsentrert buffer.

Tilsett gradvis ~1 v/v % agarose til 1x TAE buffer (~1g til 100 mL, f.eks 0.5g for 50 mL) i en flaske med volum minst to ganger volumet av løsningen, fortrinnsvis under omrøring med magnetrører - eventuelt rør for hånd med glasstav eller ved å bevege flasken.

'''OBS: Flasken må ikke være lufttett!'''

Varm løsningen i mikrobølgeovn på høy styrke til løsningen koker (ca. ett minutt). La løsningen koke i ca. 30 sekunder. Reduser varmeeffekten eller skru av ovnen dersom løsningen koker over.

Fjern flasken fra mikrobølgeovnen. Bland løsningen ved å bevege flasken '''forsiktig''' i en roterende bevegelse for hånd, eller med en magnetrører. Utvis forsiktighet for å unngå sprut. Kontroller at agarose-løsningen er klar og uten synlige partikler. Dersom det er partikler og uløst agarose i løsningen, kok løsningen igjen.

'''OBS: Bruk øyebeskyttelse! Fare for støtkoking og sprut også etter at oppvarmingen har opphørt. Flasken er varm. Bruk varmebeskyttende hansker eller annen håndbeskyttelse ved håndtering av flaske. Bruk fortrinnsvis labfrakk eller langarmede klær og unngå eksponering av bar hud. Fare for forbrenning ved søl eller sprut.'''

Tilsett DNA-fargestoff i henhold til konsentrasjonsangivelse. F.eks, for 10 000x konsentrert fargestoff, tilsett 1 uL fargestoff per 10 mL agarose løsning (5 uL for 50 mL).

Plasser et gel-støpekar på tvers av lengderetningen i et elektroforesekar og plasser en brønnkam i støpekaret. Hell varm agaroseløsning i kammeret og la stå til gel'en er størknet (ca. 30-60 minutter).

'''OBS: Elektroforesekaret skal være frakoblet fra strømforsyning når gel'en støpes.'''

Ta ut brønnkammen og snu støpekaret slik at prøvebrønnene er nærmest den sorte (postive) elektroden.

Hell 1x TAE buffer i elektroforesekaret slik at gel'en er dekket av buffer.

==Elektroforese==

*Tilsett prøvene i prøvebrønnene.
*Sett på lokket på elektroforesekaret.
*Kontroller at området rundt elektroforesekaret og strømforsyningen er tørt. Tørk bort eventuell væske.
*Koble ledningene til strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.

'''OBS: Strømforsyningen skal være avslått når ledningene kobles til!'''

*Skru på strømforsyningen.
*Juster til ønsket spenning og skru på spenningen.
*La elektroforesen foregå uforstyrret. '''Ikke rør kammeret eller ledningene så lenge spenningen er på. Dersom det oppstår tegn til lekkasje fra kammeret, avslutt elektroforesen umiddelbart uten å komme i kontakt med væsken.'''
*Skru av spenningen på strømforsyningen.
*Skru av strømforsyningen.
*Koble ledningene fra strømforsyningen. Utvis forsiktighet og bruk fortrinnsvis kun en hånd for å redusere risiko for strøm gjennom kroppen.
*Ta av lokket på elektroforesekaret.
*FLytt agarosegeleen forsiktig til en transilluminator ved hjelp av en stekespade e.l.
*Avhend agarose-gel, bufferløsning og forbruksmateriell på forsvarlig vis etter bruk. Skyll elektroforesekammer og glassutstyr med destillert vann.

Se også: https://www.addgene.org/plasmid-protocols/gel-electrophoresis/

http://www.methodbook.net/dna/agarogel.html

Typisk prøvevolum kan være 6 uL, f.eks 5 uL PCR-produkt + 1 uL 6x loading dye, eller 4 uL vann, 1 uL DNA ladder og 1 uL 6x loading dye.

=PCR=

Typiske reaksjonsvolum er 20 uL og 50 uL. Gitt en konsentrasjon i primerløsningen lik 10 uM vil i de to tilfellene tilsetting av henholdsvis 1 uL og 2.5 uL av hver primer gi en endelig kosentrasjon av hver primer lik 0.5 uM i reaksjonsblandingen.

http://dongshengbio.com/en/UploadFiles/2012516105050871.pdf

https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Typisk vil en ta ut ca. 5 uL av reaksjonsblandingen til elektroforese-analyse etter PCR.

Se også https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

==Detection of D1S80 Repeat Polymorphism by PCR==

See https://www.dnalc.org/files/pdf/forensicprofchip_d1s80_protocol.pdf

==Collection of genomic DNA by buccal swabs==

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566681/pdf/envhper00512-0045.pdf

==PTC tasting ability genotyping==

https://www.snpedia.com/index.php/Rs713598

https://www.ncbi.nlm.nih.gov/pubmed/12595690?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181941/

http://www.ncbi.nlm.nih.gov/gene?cmd=retrieve&dopt=default&rn=1&list_uids=5726

http://bioinformatics.dnalc.org/ptc/animation/pdf/ptc.pdf

==Sex determination by PCR==

See https://en.wikipedia.org/wiki/Amelogenin#Application_in_sex_determination and Fracnes et al.: Clin Chim Acta. 2007 Nov-Dec;386(1-2):53-6. Epub 2007 Jul 31.
Amelogenin test: From forensics to quality control in clinical and biochemical genomics.. (http://www.sciencedirect.com/science/article/pii/S0009898107003889)

Primers:

*AME-F: 5'-ctgatggttggcctcaagcctgtg-3'
*AME-R: 5'-taaagagattcattaacttgactg-3'

Expected results:

*If male: 1 band of 977 bp + 1 band of 790 bp
*If female: 1 band of 977 bp

==DNA fingerprinting==

===CODIS===

The Combined DNA Index System (CODIS) is a FBI program to support law enforcement by DNA-based identification. CODIS defines standards for DNA fingerprinting and a selection of genetic loci to be analyzed as part of DNA fingerprinting. The 13 CODIS core loci per 2016 are:

*CSF1PO
*FGA
*THO1
*TPOX
*VWA
*D3S1358
*D5S818
*D7S820
*D8S1179
*D13S317
*D16S539
*D18S51
*D21S11

In addition, the Amelogenin (AMEL) locus is used for sex determination.

For information on each locus, see http://www.cstl.nist.gov/strbase/

See also http://www.cstl.nist.gov/strbase/coreSTRs.htm

7 additional loci will be required from January 1 2017.

http://isogg.org/wiki/CODIS

https://en.wikipedia.org/wiki/Combined_DNA_Index_System

http://www.cstl.nist.gov/biotech/strbase/fbicore.htm

http://www.cybertory.org/resources/CODIS/

Estimated cost for CODIS primer set as described by cybertory.org, with synthesis by Macrogen Inc.: 68.2 EUR / ~75 USD / ~ 650 NOK + shipping.

===European set===

The European Standard Set (ESS) consists of the following STR loci: FGA, TH01, VWA, D1S1656, D2S441, D3S1358, D8S1179, D10S1248, D12S391, D18S51, D21S11, D22S1045

Additional Loci commonly found in European STR kits include: D2S1338, D16S539, D19S433, SE33, Amelogenin

==Colony PCR==

1. From an agar plate, select one or several colonies for colony PCR. For each colony selected, pick a small amount of colony material using a sterile pipette tip, and dissolve in 50 uL H2O. Reseed a new agar plate with 5 uL of the resulting solution(s) each in a separate spot, keeping the spots separate and noting the location of each. Alternatively, use 5 uL to inoculate a liquid culture.

Incubate the dissolved colony material at 96 C for 10 min to release DNA.

Use 1 uL of the heat-treated solution as template for PCR.

==ITS1 + ITS 4 yeast==

===Background===

See Esteve-Zarzoso et al.: ''Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers''. (http://www.ncbi.nlm.nih.gov/pubmed/10028278)

[https://www.researchgate.net/publication/13261852_Esteve-Zarzoso_B_Belloch_C_Uruburu_F_Querol_A_Identification_of_yeasts_by_RFLP_analysis_of_the_58S_rRNA_gene_and_the_two_ribosomal_internal_transcribed_spacers_Int_J_Syst_Bacteriol_49_329-337 Artikkel tilgjengelig via ResearchGate]

See also http://sites.biology.duke.edu/fungi/mycolab/primers.htm

[[Fil:ITS1 ITS4 insilico UCSC.png|miniatyr|høyre|Screenshot of in silico PCR result using the UCSC in silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr) with the primer sequences ITS1 (5' TCCGTAGGTGAACCTGCGG 3') and ITS4 (5' TCCTCCGCTTATTGATATGC 3'), selecting the S. cerevisae April 2011 (SacCer_Apr2011/sacCer3) genome assembly as the template. The result shows two expected PCR products/target regions with identical sequences. For each, the line starting with a > specifies the target location in the assembly, the target region size/expected product size, and the primer sequences, separated by spaces. The subsequent lines show the target region/expected product sequence, with the sequence of the forward primer (ITS1) and the sequence matching the reverse primer (ITS4) in capitals. The target region size/expected product size (841bp) includes the primers.]]

===PCR setup===

'''According to Esteve-Zarzoso et al.:'''

Reaction volume: 100 uL

Primer concentrations: 0.5 uM each

Forward (FWD) primer (ITS1): 5' TCCGTAGGTGAACCTGCGG 3'

Reverse (REV) primer (ITS4): 5' TCCTCCGCTTATTGATATGC 3'

(Start and end of expected product sequence = FWD primer + reverse complement of reverse primer: TCCGTAGGTGAACCTGCGG-GCATATCAATAAGCGGAGGA)

Template: Fresh yeast colony material.

Template preparation: 95 C for 15 min.

Initial denaturation: 95C for 5 min

Repeated cycles:
*Denaturation: 94 C for 1 min
*Anneal: 55.5 C for 2 min
*Extension 72C for 2 min

x 35 cycles

Final extension: 72C for 10 min

Total hold time: 190 min

Estimated program duration with OpenPCR: ~4 hr.

'''Alternative setup and reduced duration PCR program:'''

Reaction volume: 50 uL

Template: The protocol has been tested with dry yeast, see template preparation below. The protocol may also be attempted with fresh store-bought yeast, liquid yeast culture or yeast agar colony material as the template source.

PCR setup otherwise as original protocol.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 35 cycles

Step durations and temperatures are according to recommendations for Dongsheng Taq polymerase. Annealing temperature according to original protocol.

Final extension: 72 C for 10 min

Total hold time: 83 min

Run time with OpenPCR: ~ 2h 20 min.

'''Template preparation:'''

Dissolve 0.1g dry yeast (Idun tørrgjær) in 10 mL dH20. Mix well. Pipette 50 uL (-> ~0.0005g, 0.5mg yeast powder by dry weight) into a microsentrifuge tube or PCR tube and incubate at 98C for 10 minutes. Use 1-2 uL (-> ~0,00002g = 0,02mg = 20 ug yeast powder by dry weight for 2 uL) of the supernatant as template.

I tørrvekt kan det forventes at DNA utgjør i størrelsesorden 1% av cellemassen. ([http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/ ref])
20 ug tørrmasse vil da gi anslagsvis ~0.2 ug = 200 ng DNA.

DNA har molekylær vekt lik ca. 650 g/mol per basepar. ([https://www.neb.com/tools-and-resources/usage-guidelines/nucleic-acid-data ref]). S. cerevisae-genomet består av ca. 12,156*106 basepar. Basert på dette får vi da

200 *10-9 g DNA/ 12,156*106 bp) * (650 g/mol * bp) ~ 2,5*10-17 mol genomkopier.

Et mol er lik 6 * 1023. Vi har da

2,5 *10-17 mol * 6 * 1023 / mol ~ 1,5*107 (15 millioner) kopier.

S. Cerevisae har ca. 100-200 kopier av hvert rRNA-gen. Vi får da

15 millioner genomkopier * (~100 genkopier/genom) ~ 1,5 milliarder genkopier.

'''Extra reduced duration PCR program:'''

Tested 10.jul.2016. Amplification was successful for 1 of 2 samples.
Based on the results, further shortening to 25 cycles may be attempted.

Initial denaturation: 94C for 3 min

Repeated cycles:
*Denaturation: 94 C for 30 s
*Anneal: 55.5 C for 30 s
*Extension: 72C for 1 min

x 30 cycles

No final extension.

Total hold time: 63 min

Run time with OpenPCR: ~112 min. (1 h 52 minutes).

===Expected results===

'''Information from literature:'''

Expected PCR product size for ''S. cerevisiae'' (Strains CECT 1942 /ATCC 18824, CECT 1971) as reported by Esteve-Zarzoso et al: 880 bp.

'''Information from ''in silico'' PCR and published genomic sequence data:'''

[[Fil:ITS1 ITS4 S288C BLAST result.png|miniatyr|miniatyr|Graphic representation of BLAST result showing the location of the PCR target regions for the ITS1 and ITS4 primers in the S. cerevisae S288C genome (assembly R64, GenBank accession GCA_000146045.2). The query sequence is the expected PCR product sequence returned by the UCSC in silico PCR tool. While there are two target regions, there is only one expected product sequence as the sequence at and between the primer binding sites is identical for the two target regions. The locations of the target regions are shown as grey bands along the bottom, with the text "Query_185011" above each band. The left-pointing arrowing on each band indicates that the sequence of the query matches the sequence of the reverse strand as defined in the genome sequence file.]]

Expected fragment sizes as returned by in silico PCR at https://genome.ucsc.edu/cgi-bin/hgPcr (April 2011 assembly): 2 fragments of 841 bp each ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500810913_rmZAxNnexxkDaK66dHwLt86MeALA&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TCCGTAGGTGAACCTGCGG&wp_r=TCCTCCGCTTATTGATATGC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link]). The reported locations are chrXII:464259-465099 chrXII:455122-455962

The sequences of the two expected products as identified by in silico PCR above are identical. BLAST of the sequence against the R64 Assembly ([http://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/ GenBank GCA_000146045.2]) returns two matches, both on chromosome XII: Range 1: 455122 to 455962; and Range 2: 464259 to 465099. Both with 841/841 (100%) identities and zero gaps.

The genome assembly used for the in silico PCR described above is described as "Apr. 2011 (SacCer_Apr2011/sacCer3)". Although the relation or differences between this assembly/description and the R64 assembly have not been determined, the result of the BLAST search shows that the nucleotide positions on chromsome XII as reported in the ''in silico'' PCR result correspond to those of the R64 genome assembly and that there are no differences in the sequences of these regions. ([http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&QUERY=TCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAAATTTAATAATTTTGAAAATGGATTTTTTTGTTTTG%0AGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAG%0ATGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTG%0ATTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTT%0AGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTC%0AAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCT%0ACGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATC%0ATTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAAC%0AATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTT%0ATTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAAC%0ATGCGGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACA%0AATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATATCAATAAGCGGAGG%0AA&JOB_TITLE=Nucleotide%20Sequence%20%28841%20letters%29&GAPCOSTS=0%200&MATCH_SCORES=1,-2&BLAST_SPEC=Assembly&DATABASE=genomic/559292/GCF_000146045.2&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=4932&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=R&FILTER=m&WWW_BLAST_TYPE=newblast&EQ_MENU=Enter%20organism%20name%20or%20id--completions%20will%20be%20suggested&SHOW_OVERVIEW=true&SHOW_LINKOUT=true&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=true&NEW_VIEW=false&NCBI_GI=false&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&SHOW_CDS_FEATURE=false&OLD_BLAST=false direct link to search query])

(The sequence for S288C chromosome XII alone is accesible at: http://www.ncbi.nlm.nih.gov/nuccore/NC_001144.5)

[http://www.yeastgenome.org/browse/?loc=chrXII%3A463459..466018&tracks=DNA%2CAll%20Annotated%20Sequence%20Features&highlight=chrXII%3A464259..465099 Size & location of expected fragment 1, shown as highlighted area in SGD genome browser.]

[http://www.yeastgenome.org/browse/?loc=chrXII%3A454515..457224&tracks=DNA%2CAll%20Annotated%20Sequence%20Features%2CDoube_strand_break_hotspots%2CXrn1-sensitive_unstable%20transcripts_XUTs%2CScGlycerolMedia%2C3%27UTRs%2CPolII_occupancy_WT&highlight=chrXII%3A455122..455962 Size & location of expected fragment 2, shown as highlighted area in SGD genome browser]

[[Fil:RDN582-region.png|700px|sentrer|Representation of the yeast genome region centered on the RDN58-2 rRNA gene, as shown in the SGD genome browser using the latest version of the S. cerevisae S288C genome (assembly R64). The S. cerevisae genome contains 100-200 repeats of the genes shown. The yellow region represents the target area for the ITS1-ITS4 primer combination in this location.]]

The target region spanning nucleotide positions 455122 to 455962 covers the 5.8S ribosomal RNA (rRNA) gene RDN58-1 (455414..455571) and the non-coding regions ITS2-1 and ITS1-1 which flank RDN58-1. Likewise, the target region spanning nucleotide positions 464259 to 465099 covers the 5.8S rRNA gene RDN58-2 (464551..464708) and the flanking non-coding regions ITS2-2 and ITS1-2.

SGD entry for RDN58-1: http://www.yeastgenome.org/locus/S000006488/overview

SGD entry for RDN58-2: http://www.yeastgenome.org/locus/S000006489/overview

RDN58-1 and RDN58-2 are contained within the [http://www.yeastgenome.org/locus/S000029411/overview RDN1 locus]. Although the sequence included in assembly R64 only indicates two target regions for the ITS1-ITS4 primer combination, the RDN1 locus in actuality represents a 1-2Mbp repeating region containing 100-200 rDNA repeats ([http://www.yeastgenome.org/locus/S000029411/overview ref]). Thus the actual number of targets can be expected to be in this range, and some sequence variation among these is likely ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665781/ ref]).

'''Expected PCR product sequence (841 bp):''' (NB: Whitespace is included below for easier viewing, adding extra characters)

Esteve-Zarzoso et al. used the restriction enzymes CfoI, HaeIII, and HinfI to cleave PCR product DNA and perform Restriction Fragment Length Polymorphism (RFLP) analysis. The sequence below contains 3 binding sites for CfoI (GCGC) CfoI, 3 binding sites for HaeIII (GGCC) and 3 binding sites for HinfI (GANTC, where N is any nucleotide). The sequence also contains an EcoRI binding site (GAATTC, bolded).

Esteve-Zarzoso reports the restriction fragment sizes for digestion with CfoI, HaeIII and HinfI as 385 + 365 (= 750), 320 + 230 + 180 + 150 (= 880) and 365 + 155 ( = 520), respectively (fragments smaller than 50 bp not included or reported).

Based on the sequence below, the expected fragment sizes from digestion are (http://www.restrictionmapper.org/):

*EcoRI 469 + 372 (= 841)
*CfoI: 363 + 334 + 134 + 10 (= 841)
*HaeIII: 311 + 230 + 172 + 128 (= 841)
*HinfI: 362 + 355 + 116 + 8 ( = 841)

<code>TCCGTAGGTGAACCTGCGGaaggatcattaaagaaatttaataattttga
aaatggatttttttgttttggcaagagcatgagagcttttactgggcaag
aagacaagagatggagagtccagccgggcctgcgcttaagtgcgcggtct
tgctaggcttgtaagtttctttcttgctattccaaacggtgagagatttc
tgtgcttttgttataggacaattaaaaccgtttcaatacaacacactgtg
gagttttcatatctttgcaactttttctttgggcattcgagcaatcgggg
cccagaggtaacaaacacaaacaattttatctattcattaaatttttgtc
aaaaacaagaattttcgtaactggaaattttaaaatattaaaaactttca
acaacggatctcttggttctcgcatcgatgaagaacgcagcgaaatgcga
tacgtaatgtgaattgca'''gaattc'''cgtgaatcatcgaatctttgaacgca
cattgcgccccttggtattccagggggcatgcctgtttgagcgtcatttc
cttctcaaacattctgtttggtagtgagtgatactctttggagttaactt
gaaattgctggccttttcattggatgttttttttccaaagagaggtttct
ctgcgtgcttgaggtataatgcaagtacggtcgttttaggttttaccaac
tgcggctaatctttttttatactgagcgtattggaacgttatcgataaga
agagagcgtctaggcgaacaatgttcttaaagtttgacctcaaatcaggt
aggagtacccgctgaacttaaGCATATCAATAAGCGGAGGA</code>

==V9D - LS266 yeast==

===Background===
"The primers V9D (5'-TTAAGTCCCTGCCCTTTGTA-3') and LS266 (5'-GCATTCCCAAACAACTCGACTC-3') are used [to] amplify an 800-1300 bp fragment that encompasses a portion of the 18S and 28S rRNA genes and the entire intervening ITS1, 5.8S and ITS2 rRNA gene regions. These primers bind to conserved regions, with corresponding positions to Saccharomyces cerevisiae 18S (1609-1627) and 28S (287-266) rRNA genes." (Todd M Pryce. "Universal Detection and Identification of Fungi by PCR and DNA sequencing" in PCR for Clinical Microbiology, SpringerLink 2010.)

===PCR setup===

As described by Pryce (2010):

*Reaction volume: 50 uL
*Initial denaturation: 95 C for 9 min
*Repeated cycles:
*Denaturation: 95 c for 30s
*Anneal: 62C for 60s
*Extension: 72 C for 2 min
*Final extension: 72C for 5 min

x 33 (PCRS-B variant) or 35 (PCRS-D variant) cycles.

Total hold time: 136,5 min

===Expected results===
Expected fragment size: 1228 bp. ([https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=500811567_RQun5ZzNd9dZSuQJcMHIctDo3PTE&org=S.+cerevisiae&db=sacCer3&wp_target=genome&wp_f=TTAAGTCCCTGCCCTTTGTA&wp_r=GCATTCCCAAACAACTCGACTC&Submit=submit&wp_size=4000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0 direct link])

Expected fragment sizes after digestion by EcoRI: 628 + 600.

Expected PCR product sequence (S. cerevisiae):

<code>TTAcGTCCCTGCCCTTTGTAcacaccgcccgtcgctagtaccgattgaat
ggcttagtgaggcctcaggatctgcttagagaagggggcaactccatctc
agagcggagaatttggacaaacttggtcatttagaggaactaaaagtcgt
aacaaggtttccgtaggtgaacctgcggaaggatcattaaagaaatttaa
taattttgaaaatggatttttttgttttggcaagagcatgagagctttta
ctgggcaagaagacaagagatggagagtccagccgggcctgcgcttaagt
gcgcggtcttgctaggcttgtaagtttctttcttgctattccaaacggtg
agagatttctgtgcttttgttataggacaattaaaaccgtttcaatacaa
cacactgtggagttttcatatctttgcaactttttctttgggcattcgag
caatcggggcccagaggtaacaaacacaaacaattttatctattcattaa
atttttgtcaaaaacaagaattttcgtaactggaaattttaaaatattaa
aaactttcaacaacggatctcttggttctcgcatcgatgaagaacgcagc
gaaatgcgatacgtaatgtgaattgcagaattccgtgaatcatcgaatct
ttgaacgcacattgcgccccttggtattccagggggcatgcctgtttgag
cgtcatttccttctcaaacattctgtttggtagtgagtgatactctttgg
agttaacttgaaattgctggccttttcattggatgttttttttccaaaga
gaggtttctctgcgtgcttgaggtataatgcaagtacggtcgttttaggt
tttaccaactgcggctaatctttttttatactgagcgtattggaacgtta
tcgataagaagagagcgtctaggcgaacaatgttcttaaagtttgacctc
aaatcaggtaggagtacccgctgaacttaagcatatcaataagcggagga
aaagaaaccaaccgggattgccttagtaacggcgagtgaagcggcaaaag
ctcaaatttgaaatctggtaccttcggtgcccgagttgtaatttggagag
ggcaactttggggccgttccttgtctatgttccttggaacaggacgtcat
agagggtgagaatcccgtgtggcgaggagtgcggttctttgtaaagtgcc
ttcgaaGAGTCGAGTTGTTTGGGAATGC</code>

==MEATF + MEATR, animal meats==

===Background===

Lago et al. describe amplification and sequencing of the cytochrome b (cyt b) gene of several species using a set of degenerate primers. (European Food Research and Technology
March 2011, Volume 232, Issue 3, pp 509-515. http://link.springer.com/article/10.1007/s00217-010-1417-1)

===PCR setup===
Primers:
*MEAT F: CGAGGCCTMTAYTAYGG
*MEAT R: ATTGAKCGTAGGATTGCGTA

M denotes A or C. Y denotes C or T.

PCR program:

*Initial denaturation: 95C for 3 min
*Denaturation: 95 C for 30s
*Annealing: 50C for 30s
*Extension: 72 C for 30s

x 35 cycles.

*Final extension: 72C for 3 min

===Expected results===

Lago et al. report that DNA amplification with the primers MEAT F/R gave an amplicon of 555 bp in all tested species.

==PORCINE FWD + PORCINE REV, pork==

===Background===

See Ilhak and Arslan. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique. Turk. J. Vet. Anim. Sci. 2007; 31(3): 159-163: http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf

The porcine primers used by Ilhak and Arslan were designed based on Lahiff (2001). Mol Cell Probes. 2001 Feb;15(1):27-35. Species-specific PCR for the identification of ovine, porcine and chicken species in meta and bone meal (MBM).

See also GenBank accesion AF039170.1: http://www.ncbi.nlm.nih.gov/nuccore/AF039170

===PCR setup===

Primers:
*Porcine FWD: GCC TAA ATC TCC CCT CAA TGG TA
*Porcine REV: ATGAAAGAGGCAAATAGATTTTCG

*Reaction volume: 50 uL
*20 pmol of each primer

PCR program:
*Denaturation: 94 C 45 s
*Annealing: 58C 45 s
*Extension: 72C 90 s.

x 30 cycles.

===Electrophoresis===

15 uL PCR product, 1.5 % agarose at 100 V for 2 h.

===Expected results===

Ilhak and Arslan report a PCR product size of 212 bp for porcine meat.

=Lagring av bakterie-stammer i glycerol=

*1 Prepare a solution of 60 % v/v glycerol in water. (For 25 mL, mix 10 mL water and 15 mL glycerol)
*2 Add 400 uL 60 % glycerol solution and 800 uL of the culture to be stored in a cryogenic tube.
*3 Mix thoroughly!
*4 Place in 5 C refrigerator for 30 min, then move to -80 C freezer.

=Sekvenseringstjenester=

==GATC (lightrun)==
Primer specifications:
*Tm 52-58 C
*17-19 bp
*G or C at 3' end (max 3 Gs or Cs)
*maximum 4 identical sequential bp.

Preparation instructions:
*Add 5 uL template DNA (80-100 ng/uL plasmid DNA or 20-80 ng/uL purified PCR product) and 5 uL primer, 5 uM (5 pmol/uL) in one 1.5 mL tube.
*Drop off at GATC collection point or ship to: GATC Biotech AG. European Custom Sequencing Centre. Gotrfied-Hagen-Strasse 20. 51105 Köln.

==Macrogen==

Primer specifications:
*18-25 bp
*40-60 % GC
*Tm 55-60

Preparation instructions:
*Add 20 uL DNA (100 ng/uL plasmid or 50 ng/uL purified PCR product) to one tube. Add 20µl primer (10 pmol/uL) to a separate tube (?)
*Ship to: Macrogen Europe, IWO, Kamer IA3-195, Meibergdreef 39,1105 AZ Amsterdam Zuid-oost. Netherlands. Attention: J.S .Park

Preparation guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_prep.jsp

Sample submission guide: https://dna.macrogen.com/eng/support/ces/guide/ces_sample_submission.jsp

=Brukermanualer=

BioRad PowerPac Basic Power Supply: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006213.pdf