PCR machines

a_hobbs at fennel.cc.uwa.oz.au a_hobbs at fennel.cc.uwa.oz.au
Fri Feb 7 00:26:38 EST 1992


Hi,

I would appreciate some advice regarding PCR machines.

I have been using one in which the timing is controlled by a
thermocouple, that is it starts timing when the temperature in
a sample tube reaches the set temperature.  I know this isn't
very accurate but it works most of the time and gives a good
place to start for modifying times if things don't work.

Recently we bought a couple of Pharmacia PCR machines.  It wasn't clear
from the pamphlets that they work purely upon time.  Although they
supply a thermocouple, it requires external electronics or an electronic
thermometer.  It seems to me that we will then have to determine
how long it takes for samples to reach temperature for every different
set of temperatures used (ie different set of primers).  The comment
of one user was " well PCR is just a matter of trial and error anyway".

I can see that in a laboratory doing many assays with just a few sets
of primers it wouldn't matter.  But in a normal laboratory situation
it seems to me one could waste a lot of time this way.

My Questions are

1.  Does one need to calibrate such a machine each time?

2.  Does one need a permanent thermometer to monitor the machines
performance?

3.  Are incubation times particularly sensitive to other variables
such as room temperature?

4.  Is there a problem if trying to use very short annealing times
say 10 to 20 seconds?

I would appreciate any information on this topic.

Thanks in advance

Andrew A. Hobbs
Department of Biochemistry
University of Western Australia
A_Hobbs at fennel.cc.uwa.oz.au



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