Purity of DNA (Ratio of OD = 1.8-2.0) ?
rfrench at unl.edu
Tue Jan 12 10:16:19 EST 1993
eddy at boulder.Colorado.EDU (Sean Eddy) writes:
>a52041 at sakura.kudpc.kyoto-u.ac.jp (Michael CHENG) writes:
>> Hi! I need help with a question that one of the student asked me during lab
>> Why is a preparation of DNA considered to be pure if OD260/OD280 => 1.8?
>> Actually, I've never thought of it myself so I went around asking my labmates.
>>Well, nobody really knows the answer. Then I heard that there's possibly a
>>theoretical formula that sets the above value range.
>Nucleic acid absorbs with a maximum at about 260 nm. Protein, because
>of absorbance by tyrosine and tryptophan residues, shows a maximum at
>about 280 nm. (I believe phenol, another common contaminant, also
>shows a peak at around 280 nm.) Protein (or phenol) contamination
>reduces the 260/280 ratio.
>Reference: Cantor and Schimmel, _Biophysical Chemistry_, vol II;
>but any biophysical chemistry text should do.
>- Sean Eddy
>- sre at mrc-lmb.cam.ac.uk
I've always thought that OD260/OD280 ratios were rather
useless. For one thing, the molar extinction coefficients
of proteins are pretty low. It takes a lot of protein to
greatly lower the 260/280 ratio so that 260/280 values
in the 1.8 to 2.0 range can give you an overly optimistic
estimate of the purity of your nucleic acid extract. In
extracts from plants, there can be other contaminating
pigments which wreak havoc with the 260/280 number
Also, average DNA & RNA (pH 7) have an absorption
maxima at 260nm, as does AMP, TMP, and UMP. GMP
has a maxima at 252 and CMP at 271 (so does phenol!).
The spectra of C's G's, T's and U's change with pH as
well. In typical nucleic acids the absorption from C's and
G's average out to 260 but with synthetic oligos you can
get what seem to be rather strange spectra, depending
on the relative numbers of G's and C's. Thus a 260/280
ratio can be totally meaningless for oligos.
USDA, ARS, Lincoln, NE
rfrench at unl.edu
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