Vent PCR Woes

191502931 SHIVAKD at Meena.CC.URegina.CA
Thu Jun 9 22:53:46 EST 1994


I am having problems running PCR mutagenesis with NEB's Vent polymerase.
I have tried running the same reactions with Taq which work perfectly.  I have
tried varying MgSO4 conc., dNTP conc., and annealing temps all to no avail.  No
product is produced in positive controls, while the same reactions work fine
with Taq.  So my primers, template etc. seems to be working fine... what am I
doing wrong?  I am using the manufacturers suggested conditions and buffers. 
Also, a paper I found describes problems with Vent digesting unprotected
primers... is this the case and may it be my problem?  I would appreciate any
comments or tips for optimizing Vent reaction conditions or whole protocols
anyone could be bothered to post.

Thanks,
Dave Shivak



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