Bitan:Measuring DNA concentration by absorbance
Measuring DNA product by UV absorbance
The Eppendorf BioPhotometer (in Dr. Spencer’s Lab.) was used.
1. Switch ON.
2. Select dsDNA from the options, showing 1 OD260 = 50 μg/ml.
3. Add 100 ml water to the plastic cuvette appropriate for use on this machine, and press ‘Blank’. The monitor should show ‘0.000’.
4. Add 50 or 100-fold diluted DNA sample in the cuvette, insert the cuvette into the reader chamber, and press Sample. The minimum required for measurement is 50 μl.
5. Aspirate the sample off the cuvette and add the next sample. There is no need to wash between sample measurements.
6. Record the results. Calculate the actual concentration of purified DNA for the next in vitro transcription step. Take into account the dilution factor used. For reference, when A260/A280 is 1.8 for DNA or 2.0 for RNA, the sample is ultra-pure.
The same measurements can be performed in our lab using the old spectrophotometer and the Quartz cuvette. Make sure the cuvette is washed properly because the residual protein may lead to erroneous A260/A280 ratios.