OD ratios of 3 to 4!?

john brennand john.brennand at gbapr.zeneca.com
Thu Apr 18 09:35:43 EST 1996


Tom

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 
activity.  

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

John






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