1. Turn on OD absorbance spectrometer. Turn on UV source. Wait 5-10' for the UV source
2. Rinse the quartz cuvette
with dH2O. Note plastic cuvette is not
UV transparent, thus can't be used for UV measurement.
3. Adjust UV wavelength to 260 nm, fill the quartz cuvette with water. Zero the spectrometer, i.e., the OD reading for water should be 0.
4. DNA concentration from large plasmid prep is usually 0.2-2 mg/ml.
Make a 1:100 dilution of DNA in water. The final volume depends on the measurement cuvette one uses. cuvettes come in different volumes, from 0.2 ml, 0.5 ml to 1 ml (and light path length). If you suspect your DNA concentration is low, lower fold of dilution should be made.
5. Measure the OD absorbance for diluted DNA stock.
6. Repeat step 3-5 at the wavelength of 230 nm or 280 nm.
7. Calculate DNA concentration using the OD calculator of BioToolKit or using
one of the following formulas (for cuvette with 1-cm light path):
DNA concentration ( mg/ml ) = ( 49.1 x A260 - 3.48 x A230 ) * fold of dilution
DNA concentration ( mg/ml ) = ( 62.9 x A260 - 36.0 x A280 )
DNA concentration ( mg/ml ) = 50 x A260 * fold of dilution
For single-stranded DNA or RNA
Concentration ( mg/ml ) = 40 x A260 * fold of dilution
8. Turn off UV source and the spectrometer.