PCR Question

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Feb 16 11:06:25 EST 1995


Jim Kyriannis asks:

> I have 2 30-mer PCR primers which have a Tm higher than 100 degrees.  I
have tried to use these two primers to PCR directly from yeast colonies with no
success.  I have used other primers under the same conditions, which have
worked just fine.  Could the high Tm be the source of the PCR reaction
failures?

I suspect that the Tm over 100 degrees is an artifact of using some equation
that predicts Tm in 1 M salt.  However the Tm of a 30 mer in PCR buffer may
well be too far above the annealing temp. that you are using (which has to
be <= extension temp.) and so your primers would be subject to increased
false priming because of the non stringent conditions.  The usual failure
mode in this case would be too many bands, not failure to make the correct
band.  Your primer design method has failed badly on suggesting the appro-
priate length of primer or annealing temp.; 
hence I have to suspect that it also failed to
detect problems related to hairpinning, primer dimer formation, etc.
These problems give failure modes with no bands.  

When I first started doing PCR, I thought you could just rip off a primer
with 20 or so bases and that they'd work so long as the 3' ends didn't
have obvious primer/primer complementarity.  This just led to a circus
of failed rxns and PCR artifacts.  Take some advice from the war-weary
and invest in some software to design good primers and spend some time
learning about the nuances involved.  Start by looking around your 
institute for such a package and then absorb everything you
can from the manual about what it's trying to accomplish.

Good luck.

Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu







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