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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