PCR anomaly

Leman leman at leman.org
Tue Oct 12 10:43:12 EST 1999


On Tue, 12 Oct 1999 09:53:49 -0400, Mark Edward Bowen
<mb at laplace.csb.yale.edu> wrote:

>My PCR reactions have suddenly become dominated by two well defined and
>reproduceable small MW (~500bp) products.  It happens with different
>buffers, enzymes, primers and templates.
>[...]

Here is the answer, already stated in your question: a component you
didn't mention is water. H2O is the first and most common source of
contamination, since it usually sits in a bigger bottle/tube at the
room temperature. I had a similar problem once. Now it is the rule --
throw away your H2O every Monday and pour (not pipette!) new portion
in a sterile Falcon tube from an autoclaved bottle. Yet another
component you didn't mention is your dNTPs solution. I assume you use
the same stock with different buffers, enzymes, primers and templates.
It's a good idea to mix dATP, dTTP, dGTP and dCTP stocks in one common
"dNTP" stock (without forgetting to divide their concentration by 4 in
your future calculations ;-)) and make 20-50 µl aliquots to store at
-20C. This way you don't use the same stock tube more than once or
twice.
Good luck.

	Leman



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