PCR with Pwo DNA polymerase from Boehringer

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Thu Dec 12 23:34:16 EST 1996


If the Barnes paper isn't part of the FAQ list, it ought to be.
(WM Barnes. PCR amplification of up to 35-kb DNA with high fidelity and
high yield from lambda bacteriophage templates. PNAS 91: 2216-2220
(1994).)  The basic idea is that you use a mixture of klentaq and a
proofreader (Pfu or Pwo) at a ratio of 250U KT to 1U proofreader. 
This gives the same fidelity as the proofreader alone, with a much
better yield than the proofreader alone.  The key is that klentaq is
poor at extending off mismatches (errors) (unlike taq which is much
better at extending off mismatches).  The trace amount of proofreader
functions to trim back the mismatches and allow klentaq to extend
further (and therefore the proofreader doesn't chew up your primers). 
Works as advertised in my hands.  Gotta use the KCl free buffer
mentioned in the paper, though.  It works really well, even on tough
to amplify sequences. (BTW I use 20mM tris HCl pH 9.0, 15mM (NH4)2SO4
3.5mM MgSO4 final for mine).  Good Luck.

					Regards,	SVEN


In article <32B09D66.69A1 at brosh.cc.biu.ac.il>, "Don J. Katcoff" <katcoff at brosh.cc.biu.ac.il> writes:
> We have been trying to use Pwo DNA polymerase from Boehringer for PCR, 
> but have been having difficulties.  Using the same template and primers 
> but other taq polymerases we have no problem.  We are interested in 
> particularly high fidelity.  Any help would be appreciated.
> 
> Don Katcoff
> katcoff at brosh.cc.biu.ac.il



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