A260 or Fluoromete ??? Which is correct

john brennand john.brennand at gbapr.zeneca.com
Tue Mar 19 11:16:49 EST 1996


Shaun

We have seen the same sort of discrepancies as you.  I have always put 
it down to the DNA being contaminated with very small nucleic acid 
fragments and nucleotides, that come down in the precipitation (hence 
the RNAse treatment will exacerbate the effect by creating more, smaller 
molecules). These do have a large absorbtion and of course you cant see 
them very well on a gel.  Back in the old days of Maxim Gilbert 
sequencing they were a real problem as they tended to "suck up" your 
label, whereupon they became obvious on the gel.

CsCl banded plasmid was always bad in this respect whilst Qiagen 
purified plasmid doesn't seem to suffer from this as much.

We now routinely double check by 1st checking A260, and then running 1 
ug of the DNA on an agarose gel and eyeballing the intensity compared 
with a bit of DNA of similar size who's concentration you are happy 
with.

John







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