Vent PCR Woes

Olav Hungnes ohungnes at bioslave.uio.no
Tue Jun 14 05:11:31 EST 1994


191502931 (SHIVAKD at Meena.CC.URegina.CA) wrote:
: 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
Some time ago I did Vent PCR mutagenesis, and a proportion of my products 
had lost the mutation, presumably due to primer degradation past the 
mutation site. The tech.bulletin for the enzyme stated that it could 
degrade free primers down to about 15-mers, I think. You can perhaps 
compensate for some degradation by lowering the annealling temp., but 
then you wouldn't want to allow primers degraded beyond your mutation to 
anneal, so don't overdo it. I never had any problems getting the enzyme 
to work, but it didn't give as much product as Taq.

Good luck,
Olav

-- 
_______________________________________________________
Olav Hungnes                     ohungnes at embnet.uio.no
National Institute               Phone  (+47)22042200
of Public Health                 FAX    (+47)22353605
Oslo, NORWAY
_______________________________________________________



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