multiple primers in PCR

John Ladasky jladasky at cmgm.Stanford.EDU
Wed Nov 26 15:21:54 EST 1997


In article <qtang-2611971253360001 at qtang.utmem.edu>,
sanglier <qtang at nb.utmem.edu> wrote:
>I'd like to use three different primer pairs to genotype my heterozygous
>double-mutant mice. The oligos are all roughly 20mers with similar Tm, and
>don't look like they will form dimers from a cursory check. The products
>are between 200 and 500bp. My question is, what concentration of each
>primer should I use? I had tried to use 1/3 of each to keep the final
>primer concentration the same as with single primers but had cruddy
>results. Is there anything else I should look out for? 
>
>I'll be grateful for any suggestions!
>Q

	I don't know what your original primer concentration was, but my
guess is that you are using way too much primer.  When you use too much
primer, you will frequently, but not consistently, inhibit one or more
of the reactions you want to see.  In many cases, before I knew better,
I added two working PCR's together and got no bands!

	In my experience, multiplex PCR's work best with roughly 10-fold
less primer than I was originally taught to use in single-locus PCR.  My
original references taught me to add primers to a final concentration of
about 1.0 uM (each).  The multiplex reactions that I've designed work at
concentrations of about 100 nM/primer.  This is just a rough concentra-
tion.  You will usually need to balance the amount of each primer so that
each product is amplified with equal intensity.  Can you perform each
PCR by itself in a separate tube at the same temperature?  That is an im-
portant first step.

	There's a recent, long-overdue Biotechniques article that covers
issues related to multiplex PCR pretty well.  O. Henegariu et. al., 
September 1997 Vol. 23 No. 3, pp. 504-511.  You can also check out Dr.
Henegariu's web page at http://medgen.iupui.edu/~ohenega/PCR.html.

	Good luck!

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.						- John Ladasky



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