Long PCR

Bryan L. Ford fordb at bcc.orst.edu
Sat Oct 4 19:13:01 EST 1997


Albé van der Merwe ZA wrote:
> 
> Rajesh Kumar <rk6n at virginia.edu> wrote in article
> <3432715B.72BA at virginia.edu>...
> > Hi,
> >
> > I need to do a long PCR (~9 kb). Can somebody suggest me a suitable
> > polymerase and a source of it ? How about using a mixture of two
> > polymerases, say, Taq and Pfu ?
> >
> > Any suggestion will be highly appreciated.
> >
> > Rajesh
> >
> 
> Try Expand mixture. It has Pwo, and Taq (I think). Annealing temp. should
> be 68*C for >3 kb, and 72*C for <3 kb. Hope this helps.
> 
> [:)

Friend:

I think there is a bit of a slip here. The *extension* temperature
should meet the above parameters. This would then correspond with Wayne
M. Barnes originally published long PCR findings (Proc. Natl. Acad. Sci.
USA Vol. 91, pp 2216-2220, March 1994) that long accumulated time above
68 degrees increases likelihood of DNA damage and thereby decreases the
likelihood of intact template and successful long PCR. Annealing
temperatures are determined largely by the base composition and length
of the primers as in usual PCRs.

Sincerely,
Bryan




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