RNA extraction

David F. Spencer dspencer at is.dal.ca
Tue Jul 25 11:18:45 EST 2000


In article <rh-2107001554340001 at a6-curtis.ibls.gla.ac.uk>, Robert
Hartley <rh at mblab.gla.ac.uk> wrote:

> One thing that als oaffect the spec is the pH of the water you are using
> for resuspending.

That is technically correct but probably not a factor in the way you
propose.

Good quality distilled water exposed to air will become saturated with
CO2 and its pH will drop to about 5.5 from the carbonic acid. The
nearest pK (below pH 7) in nucleic acids is for the exocyclic amino on
carbon 4 of cytosine/cytidine which has a pK (for protonation) of 4.5
to 4.6. So unless there is a significant amount of acidic buffer
contaminating the original RNA solution the final dilution of the RNA
into the water in the cuvette should give pH's above 5-5.5 and thus the
C's pK wouldn't be relevant.

Of course the Trizol extraction mix is quite acidic (the pH could be as
low as 4) so if there were much of this carried over into the final RNA
solution (caused by inadequate washing of the pelleted RNA or by doing
one rather than two ethanol ppt'ns) then the pH in the cuvette could be
low enough to cause problems. Protonated 'C' has an absorption at 260
nm that is nearly the same as at neutral pH but the absorption at 280
nm is much higher in protonated 'C' compared to neutral 'C'.  That
would cause the 260/280 ratio to be lower than desirable (and mimic the
effects of protein contamination).  A second ethanol ppt'n (using
untitrated/neutral sodium acetate) would solve that problem.

Dave






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