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
- 1 Liquid handling
- 2 Thermocyclers
- 3 DNA quantification
- 4 Centrifugation
- 5 Weighing
GeneQuant Manual: http://www.americaninstrument.com/pdf/1976E-DNA.pdf
- Light path height = 15 mm
Eppendorf biophotometer 6131
- 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)
- Photometric random error: <= 0.005A at 1A.
- Photometric systemic error: +- 1% at 1A.
Height of light beams in cuvette: 8.5 mm
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.
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
- 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 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/
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."
- 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
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