PCR: Why is my TAQ unfaithful

[David J. Meyer]

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!

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