PCR: Why is my TAQ unfaithful

neale at mbcf.stjude.org neale at mbcf.stjude.org
Wed Nov 23 12:25:14 EST 1994


I tried mailing this direct to you, but encountered mail errors. So...........

Julie,

While I can't say why you have suffered so many mutations with your PCR, I can
say that we have experienced similar problems when using the "regular" Thermus
aquaticus polymerase for cloning. Tour frequency of mutation seems
extraordinarily high!

The way we got around this was to use the Pfu polymerase (Stratagene, I
believe) which has proof reading capacity. This greatly reduced the infidelity
of the PCR and we have not yet found a clone with a mutation using this
polymerase. (Our PCR product was also small--around 500bp)

Hope this helps,     

Regards,

Geoff Neale

Dept. of Virology and Molecular Biology        Internet: neale at mbcf.stjude.org
St. Jude Children's Research Hospital             Phone: (901) 522-0400
Memphis, TN                                         Fax: (901) 523-2622

 

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



More information about the Methods mailing list