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(→‎Eppendorf biophotometer 6131)
Linje 22: Linje 22:
  
 
*Device: Single-beam filter photometer with reference beam and fixed wavelengths.
 
*Device: Single-beam filter photometer with reference beam and fixed wavelengths.
−
*Light path:
+
 
  
 
Cuvette types (min volume):
 
Cuvette types (min volume):
Linje 31: Linje 31:
  
 
Wavelengths: 230,260,280,320,562,595
 
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
 
Height of light beams in cuvette: 8.5 mm
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See also: http://www.biocompare.com/Application-Notes/138277-Reproducible-photometric-determination-of-DNA-concentrations-using-the-Eppendorf-UVette-in-the-Eppendorf-BioPhotometer-plus/
 
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===
 +
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 DSBio NF water.
 +
 +
100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.
 +
 +
50 uL of a solution of ITS1 DNA single-strand 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)
 +
 +
Complete results: 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: 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.
 +
 +
 +
 +
Note 1: The 10 uM solution was previously prepared 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.
  
 
=Centrifugation=
 
=Centrifugation=

Revisjonen fra 7. aug. 2016 kl. 19:22

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

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 DSBio NF water.

100 uL NF water in another cuvette was measured as a control. Result = 0.000 ug/uL.

50 uL of a solution of ITS1 DNA single-strand 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)

Complete results: 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: 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.


Note 1: The 10 uM solution was previously prepared 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.

Centrifugation

Eppendorf 5415D

Manual: http://www.nist.gov/ncnr/upload/Eppendorf_5415R_Centrifuge_Manual.pdf

  • Max speed: 13 200 rpm
  • Max centrifugal force: 16 110 rcf
  • 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
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