Vent PCR Woes
hdang at cns.neusc.bcm.tmc.edu
Mon Jun 13 10:36:37 EST 1994
You didn't do anything wrong, to my experience. I tried Vent and DeepVent, never
got any decent PCR. Stratagene's Pfu worked a little better, but nothing to really
depend on. One thing you can try if you have to have them work, is 10% glycerol.
Apparently, 3'-5' exo proofreading activity makes them very "picky", so there is
one more enzyme you can try, Ultma (Perkin-Elmar) has a reduced exo activity which
allows it to be more efficient, so I heard, but it is just like mixing Taq and an
axo enzyme (Barnes' paper).
I assume you are inverse-PCRing for mutagenesis and need blunt ends. To that
purpose, Barnes' way and Ultma will probably fare the same, lots of 3' tails of
As. This is what I do: PCR with Taq/exo mix or Taq alone, check products on a gel,
add Pfu afterwards in the same tube and "polish" the ends, like 72C, 30 min.
Incidentally, the Stratagene rep told me just last Fri that they are using the
same thing to increase their blunt end PCR cloning kit efficiency, and probably
will packaged it in some way, kits for $$.
If you are interested in my detailed protocol, drop me a line.
hdang at channel.neusc.bcm.tmc.edu
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