HELP! VENT / Pfu amplification

Dan Baggott dbaggott at reed.edu
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



More information about the Methods mailing list