Vent PCR Woes
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.
: 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.
Olav Hungnes ohungnes at embnet.uio.no
National Institute Phone (+47)22042200
of Public Health FAX (+47)22353605
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