Just a couple quick comments regarding PCR at higher altitudes and enzyme
stability. Taq polymerase has a temperature optimum, according to Perkin-
Elmer, of 75-80?C. The half-life of the enzyme though drops off sharply above
94?C (92.5?C/>130 min, 95?C/40 min, 97.5?C/10 min). The half-life of Taq can
be extended at 95?C with the addition of 10% glycerol to the reaction
mixture, but this may or may not affect the PCR. The real concern with PCR at
high altitudes and not being able to reach temperatures higher than 89?C is
the denaturing of the DNA strands which is necessary for further cycles of
PCR. I think the addition of DMSO, as already mentioned by several others, is
a good idea. After the initial denaturation of your template DNA (boiling in
well sealed tubes?), would the denaturation of the PCR products, which would
be much smaller in length relative to the initial template DNA, be easier to
accomplish, ie. sufficient denaturation of PCR products at lower temperatures?
I'm curious to find out what works.
David Juck
McGill University, Macdonald College
Montreal, Canada
------------------------------------------------------------------------------
------------------------------------------------------------------------------
Dear Netters:
A quick observation (somebody correct me if this is not accurate):
Taq is an enzyme, albeit one with a very high temperature optimum.
Therefore, at a temp slightly below boiling in Quito (89 C, was it?),
shouldn't Taq still work, even if slightly less than optimally, such that
routine amplifications could still be accomplished? Does anyone know how
rapidly Taq's efficiency falls off either side of 94 C? I assume the dropoff
is steeper above 94 C?
|===========================================|
David A. Watson, Ph.D. (dwatson at plains.nodak.edu)
Asst. Prof. of Veterinary and Microbiological Sciences
North Dakota State University
---------------------------------------------------------------------------
"Some are wise, some otherwise."
|===========================================|
