PCR: Why is my TAQ unfaithful

Kai-M. Toellner K.M.Toellner at bham.ac.uk
Mon Nov 28 13:01:01 EST 1994


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

The Taq buffer supplied with Promega Taq Polymerase has pH 9.0, which is 
rather high compared to the usually used Taq buffer, which has pH 8.3.

In
  TI: HIGH FIDELITY DNA-SYNTHESIS BY THE THERMUS-AQUATICUS DNA-POLYMERASE
  AU: ECKERT_KA, KUNKEL_TA
  JN: NUCLEIC ACIDS RESEARCH 1990 Vol.18 No.13 pp.3739-3744
was shown, that the error rate of Taq-polymerase rises 60-fold, when the 
pH is increased from pH 5-6 to pH 8.2. I assume, the error rate will 
rise further, when the pH is increased to pH 9.0. 
So try another source for your Taq or carry on with the cheap Promega 
stuff and ask PE for some tubes of buffer;-)

Good luck, Kai



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