I ran across this in Molecular Cloning 3rd edition volume 3 page A8.20
"Note, however, that the extinction coefficients of both DNA and RNA are affected by ionic strength and the pH of the solution (references). Accurate measurements of concentration can only be made when the pH is carefully controlled and the ionic strength of the solution is low."
May help explain the variation that I have seen.
On Thursday, February 7, 2013 11:36:36 AM UTC-5, shamrock wrote:
--I take DNA concentrations as determined via spectrophotometer with a bit of skepticism and verify by electrophoresis.If I had some known standards that would validate the calibration of the spec I might have more confidence in the readings, but I've often seen discrepencies between the spec and electrophoresis results.
On Wednesday, February 6, 2013 9:28:02 PM UTC-5, Josh Perfetto wrote:Hi,I'm wondering what the expected accuracy of UV spectrophotometers are when quantifying dsDNA. I had DNA in TE which I believed was 100 mg/ml based on resuspending a known quantity of DNA as measured by a synthesis company. I dilluted it 50x to a total volume of 100ul and measured in two different spectrophotometers.Spec 1 (Thermo Electron Helios Gamma):A260 = 0.031 AA280 = 0.019 ACalculated concentration: 77.5 ug/mlSpec 2 (Eppendorf BioPhotometer):A260 = 0.050 AA280 = 0.022 ACalculated concentration: 125 ug/mlThe consistency was a little disappointing. Is this normal or do you think there is a problem with my specs? I suppose I could get more accuracy by diluting less but I hate using up so much plasmid just for quantification.-Josh
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