HELP! VENT / Pfu amplification

Dan Baggott dbaggott at
Wed Jun 26 18:45:33 EST 1996

C.Pical wrote:
> Yes, I have had this problem, using Pfu. I managed to get some
> amplification, by using tha reaction buffer provided with Taq, and
> doubling the extension time (Pfu has proof-reading activity). Still,
> the yield was poor compared to Taq (about 20% I would say). But that
> was enough for successful cloning. I have not tried ro increase the
> yield, by playing with MgCl2,... because Pfu is expensive. Still, I
> think there must be a way to get good yields. Any other suggestions
> anyone?

I didn't see the post to which you're replying but am curious:  are you
suggesting that you need to double the extension time when using an
enzyme that has proof-reading activity?  I've never used Pfu but have
used Vent (which also proof-reads) and, at least for Vent, you certainly
do not have to double the extension time.  In fact, I would have thought
that if your enzyme has 3'->5' exonuclease activity you'd want to be
extra careful that the extension times weren't too long otherwise the
enzyme would start chewing things up.

Dan Baggott

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