OD ratios of 3 to 4!?
john.brennand at gbapr.zeneca.com
Thu Apr 18 09:35:43 EST 1996
15 or so years ago someone explained it to me thus:-
Take a solution of "pure" DNA - check the A260, then change to A280 and the
value should be 1/2 that value (ie a ratio of 2).
This is because, if you look at the spectrum (ie do a simple wavelength scan
190-320) of the solution, 280 nM is the point which intersects 1/2 way up the
peak as the absorbtion trails off.
Now, as the protein absorbtion max happens to be at 280, any dimunition in the
260:280 ratio is likely to reflect protein contamination. The exact level of
which can be read off a Nomograph (I used to have one of these but it has
disappeared and I don't know where the original came from).
Thus, it follows, that an INCREASE in the 260:280 ratio must be due to
contamination from molecules that absorb more at 260 than 280 - either because
of a leftward shift in the spectrum or a tighter absorbtion spectrum.
My bet is that your plasmid will be contaminated with nucleotides, or small
oligonucleotide fragments or some other organics, that have this spectral
I used to get similar problem in the bad old days of CsCl preps when we found
that the DNA was useless for end labelling prior to Maxim-Gilbert sequencing,
the small fragment contaminants just sucked up the label !
Since moving to column chromatography some 10 years ago (pre-Qiagen!) we have
never observed this phenomena again
Either that or your Spec. is knackered !
Hope this sheds some light
More information about the Methods