PCR: Why is my TAQ unfaithful

[Duncan Clark]

In article: <3bd5st$2tp at sun4.bham.ac.uk>  K.M.Toellner at bham.ac.uk (Kai-M. Toellner) writes:
> 
> 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. 

That is all very well but the buffer pH will fall dramatically at 70C 
from the pH 9.0 at 25C. If you look at Wayne Barnes's papers on LA PCR 
you will find that higher pH is better and fidelity remains very high. 
As to the original post with the 3errors per 180bp this is truly 
appalling. I never amplify less than 2-2.5kb and use a buffer with pH 
8.8. I express everything I PCR and only rarely DON'T get expression of the 
full length proteins. I'm sure I have errors but if so they are silent or 
non-lethal for my purposes. To further improve fidelity try adding 
1/64 unit Pfu (Taq extender!) per 2.5u Taq. Also does your template have 
really bad secondary structure or 'cos that may be the problem.

Good luck.

Duncan     

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