Trouble finding DNA concentration on Spec..

Andy Phillips andy.phillips at bbsrc.ac.uk
Wed Feb 14 18:29:22 EST 1996


Charles A Miller wrote:
 
 >         I am am having trouble determining DNA concentrations on a
 > spectrophotometer. When I measure the UV absorbance, I consistantly get
 > 0 or even a negative number when it is obvious through gel
 > elctrophoresis of the supercoiled plasmid that there is plently of DNA.
 
For nucleic acid quantification, I don't think It's really safe to take a 
single reading at 260nm. There are plenty of things that can absorb in this 
region - EDTA has a huge absorbance in the far UV and is still significant at 
260nm. I always run a scan from about 220 up to 300 nm. That way you can tell 
if the absorbance at 260 is part of a background absorbance from a buffer 
component or contaminant, or a proper nucleic acid peak. You should also zero 
the machine using the same buffer that the DNA or RNA is dissolved in. I 
always dilute the DNA sample into water, and first run a background scan of 
the same amount of buffer in water. 

If you aren't getting any OD at all, first try calculating the OD that you 
would expect from a rough estimate of DNA concentration. You may find that 
the dilution you're using yields an OD that's below the sensitivity of the 
spec. 

eg. A 0.1mg/ml DNA solution diluted 1000-fold in water will have an OD260 of 
about 0.004 - out of the range of some spectrophotometers.

Andy
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