DNA Quantification Procedure
- Pipet 125ml of dH2O into cuvette (path length: 10.00mm) to be used.
- Set spectrophotometer for dsDNA measurement, then wavelength for 260 nm and 280 nm by following instructions in instrument manual. Insert cuvette.
- Zero spectrophotometer using cuvette containing dH2O as a blank, then set Blank button
- Sample dilution in the measuring cuvette can be entered using the dilution Key before the measurement of samples begins and then include automatically in the result calulation. Enter dilution-> enter sample (for example 2 ml) and diluent (123 ml) volume.
- Pipet 2 ml of the sample into a 1.5 ml microtube containing 123 ml dH2O. Mix DNA by vortexing.
- Remove dH2O from blank cuvette with a Pasteur pipet.
- Pipet sample into cuvette, then set Sample button. The concentration of DNA is displayed. The dilution factor entered remains stored for the calculation of further sample results until it is overwritten.
- Remove sample from cuvette with a Pasteur pipet. Wash cuvette by rinsing with dH2O several times or rinsing with the cuvette washing device. Replace cuvette in holder.
- Repeat process Blank- Sample to all other sample to record ODs (see below) for all samples.
For calculation of DNA concentration in samples free of RNA, the following conversion factor is used: 1 OD260 = 50 mg of DNA/ml.
DNA concentration in mg/ml can be calculated as follows:
OD260 x 50 mg DNA/ml x Dilution Factor
mg DNA/ml = ___________________________
With a dilution factor of 62.5 (i.e., 2 ml in 123 ml), this formula reduces to:
mg DNA/ml = OD260 x 3.125
OD260/OD280 =1.7 -1.8
- Molecular Biology