PCR: Why is my TAQ unfaithful

David J. Meyer meyerdj at biovx1.biology.ucla.edu
Wed Nov 23 18:33:58 EST 1994

In article <jwilber-221194142007 at jwilberding.chem.nd.edu>,
jwilber at pop.nd.edu (Julie Wilberding) wrote:

> Hi,
> I have been tying to PCR three constructs.  I use Promega TAQ, 100uM
> dNTP's, buffer and the proper amount of primers and template (Double
> stranded circular plasmid).  My mutation rate is high ( 3 in one 180 bp
> fragment).  I have changed dNTP, TAQ, with no change.  This is a problem
> recently encountered in our lab.  The PCR conditions are 94 1 min., 55 1
> min., 72 2 min., 30 cycles with a final cycle b3ing a 10 min extension at
> 72.  Is the circular template a possible cause???  Efficiency is good but
> fidelity sucks!  Any help or advice would be greatly appreciated.
> Julie

Hi, Julie!

   I've probably sequenced about 3 kB of PCR-produced constructs from ds
plasmid DNA (mutagenizing within one of the oligos only‹hopefully), and
never found an error. What I do is to titrate plasmid (template)
concentration and cycle number vs. yield, then use the conditions which
give me *just* enough product to clone. I don't know exactly what your
conditions are, but you may reach this product yield after only 15 cycles
and plateu at 20 cycles, then spend the next 10 doing mutagenesis!


David J. Meyer
Department of Biological Chemistry
University of California-Los Angeles
meyerdj at biovx1.biology.ucla.edu

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