From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995
Path: biosci!PLAINS.NODAK.EDU!dwatson
From: dwatson@PLAINS.NODAK.EDU (David Watson)
Newsgroups: bionet.diagnostics
Subject: High-altitude PCR
Date: 24 Mar 1995 08:21:16 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
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One more bit re: the PCR question. Tm's for most DNA's of approx. 40 % G+C
are 85-90 C (in, e.g.  TE buffer), such that with even 40-50% of the
template melted, it might still be possible to routinely amplify targets by
melting at say 88 C.  I think David Juck makes a good point when he says
that subsequent denaturation of the much smaller amplicon could be much
easier (but only if it doesn't have a high G+C content).  Before adding DMSO
or glycerol or whatever, I'd be inclined to just give it one shot at 88 C.
You might accomplish your objective without further fine-tuning.  There
might also be the added benefit of less temperature-related damage to the DNA.

Dave Watson


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