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
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 rcf.
- 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
- Dimensions: Height 23 cm, Depth 31 cm, Width 23 cm