DMSO in PCR reactions

z z-suldan at ski.mskcc.no.spam.org
Wed Dec 3 08:08:31 EST 1997


In article <881142698.8533 at dejanews.com>, bia49 at cc.keele.ac.uk wrote:

> We are attempting to amplifiy a PCR fragment of about 990bp.  As our
> template is very GC rich we have included DMSO in the reaction buffer. 
> (We tried formamide, with little success)  At 2% DMSO ,the fragment we
> get is about 1.2kb, whilst at either 6 or 10% DMSO, we get a fragment of
> the correct size.  Can any one suggest any reason for the discrepancy in
> band sizes? Logic seems to suggest that if the DMSO is affecting the way
> that the DNA migrates in the gel then the samples with the higher
> concentrations of DMSO should be most affected, but it is in fact those
> with the lower concentrations which are giving the larger than expected
> fragments. Please E-mail replies to bia49 at cc.keele.ac.uk
> 
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Interesting question. Off the top of my head, it sounds like there is a
second priming site for one of the primers. The 2% DMSO allows the
polymerase to read through the GC rich region, but doesn't destabilize the
incorrect priming enough until you go up to 6-10% DMSO. Do you have
sequence info for the surrounding region? Have you checked for other
regions with high homology to your primers?

As for DMSO affecting the migration through the gel, I haven't seen it
with my samples. If you're worried, try adding an equal amount of DMSO to
your positive control PCR rxn and/or add it to the MW markers. To my mind,
those two should control for effects on migration.

Good luck,

Zal



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