The most common method for quantifying DNA samples is by conventional absorbance measurements; nucleic acids have an absorption maximum at 260nm. Most samples contain contaminates such as proteins and single stranded DNA/RNA that absorb maximally at 280nm. The equation for calculating DNA in the presence of contaminates is:
A /A = pure dsDNA
The higher the ratio, the more pure the DNA sample. It is acceptable to have a ratio between 1.8 and 2.0 for a cuvette spectrophotometer.
Comparing Results of a Spectrophometer and a Microplate Reader Absorbance is defined by Beer-Lambert equation
where ε= molar coefficient, b= pathlength and c=concentration. When the molar coefficient and pathlength are constant, absorbance is proportional to the concentration.
For a standard cuvette reader, the pathlength is usually defined as 1 centimeter. Therefore, with a conventional absorbance reading an A260 of 0.1 O.D. corresponds to 5µg/mL dsDNA solution. Because of the shorter pathlength in a microplate reader, this value is somewhat smaller (0.07 O.D. corresponds to 5µg/mL dsDNA solution).
Please click here for DNA Quantification using spectrometer-based instruments (FLUOstar / POLARstar Omega).
The BMG FLUOstar and POLARstar Galaxy are able to perform multichromatic absorbance assays. The instrument must have a low UV optical system installed (top and bottom optics). The filters, 280-12nm and 260-12nm, should be installed on the excitation side; there should be an empty position on the emission side. Install the absorbance optic with the connection to the excitation positioning wheel and the turn the emission positioning wheel so that the bottom optics are in the 12 o‘clock position.
In the Setup menu, select Reader Configuration and set the instrument for absorbance. Check the Filter menu to be sure that the 280nm and 260nm are in the correct position.
Click on the test setup icon. Select the following parameters:
- Plate mode
- No. of cycles: 5
- No. of flashes: 20
- Positioning Delay: 0.5 s
For number of multichromatics, enter ‚2‘ then click on the arrow; the Multichromatics window appears.
Enter the first filter pair: Excitation 260nm; Emission ‚empty‘
Enter the second filter pair: Excitation 280nm; Emission ‚empty
Before starting the measurement, perform a gain adjustment for each filter.
Open the Excel evaluation software and double click on the desired testrun. On the Raw Data page toggle the Calc. Start and Stop windows so that all cycles are integrated (cycles 1 to 5). In the calculation pull-down menu select ‚Average‘. Press the Update button.
Open the Evaluation96 worksheet. Setup the three tables in the following manner:
Raw data-blank; select ‚1‘ in the multichromatic box. The data from A260 will be shown on the table.
Raw data-blank; select ‚2‘ in the multichromatic box and the data from A280 will be shown.
Select Raw data-blank. In the pull-down menu on the right, select Table1/Table2. This is the ratio A260/A280.
The following data was generated using a Costar low UV plate. The DNA source was lambda phage DNA from Molecular Probes (100µg/mL). The DNA underwent a 2-fold serial dilution using a starting concentration of 50µg/mL. The dilution buffer was 1xTE.
The acceptable ratio (in a microplate reader) for dsDNA is in the range of 1.8 to 1.4. A ratio of 1 implies that the sample is not pure enough.