An overview of available equipment and equipment of interest.
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
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)
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.
- 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