Annealing Temperatures for PCR

Brian Foley brianf at
Sun Aug 14 07:01:26 EST 1994

rosas at wrote:
: Hi

: Primer       5'   cttctttcagctggattctaatttaaag    3'
:                          --

: I have already done some PCRs using different ATs, but all of
: them have given me very low yield of my PCR product...

    I think you'll be making a significant amount of product
from the following hybridization:

Primer       5'   cttctttcagctggattctaatttaaag    3'
Primer            3' gaaa                 tttcttc 5'

	This will add an extra AAG to the 3' end of the primers
in early rounds.
	Now you'd think that just a few bases of anealing at
the 3' end would not work...  But it does due to the 
very high concentration of primers in the early rounds.
	Perkin-Elmer used to include a set of "test" primers
with their PCR kit which had only a 2 base complementarity
at the 3' end in order to show how much "primer dimer"
such a problem can cause.

	If you are making any such product, it may not appear as
a "primer-dimer" band on a gel, because it will not be a true
full length ds-DNA primer dimer, but rather will be single stranded
and jut have 3 extra bases (AAG) on its 3' end.

	You could start with low level of this primer and
then add in more after 8 or 10 cycles, when the concentration
of the template has increased so that the ratio of primer
to template is not so high.  I've never tried this so
I don't know if it will work, but "in theory" :)  it should.

*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *

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