PCR: Why is my TAQ unfaithful

[Marc Goldstein]

Subject: Re: PCR: Why is my TAQ unfaithful
Newsgroups: bionet.molbio.methds-reagnts
References: <jwilber-221194142007 at jwilberding.chem.nd.edu>
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Julie-

	I recommend switching to a polymerase that has 3' to 5' exo 
proofreading activity, such as the Pfu polymerase.  After sequencing 
about 6 kb worth of TAQ-amplified products, and finding about 1-2 errors 
per kb, I switched.  I then sequenced close to 10 kb without finding a 
single error.  One thing to note: Pfu has a lower processivity than does 
Taq, so you need to increase incubation times or enzyme concentration a 
bit.  Also note that it doesn't have the terminal transferase activity, 
so you can't go directly into a TA vector, although there are easy ways 
around this.

Good luck,

Marc Goldstein, PhD
Section of Plant Biology
UC Davis
magoldstein at ucdavis.edu

Julie Wilberding (jwilber at pop.nd.edu) 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