HiFi PCR and RE:TritonX100

Rich Dudley rdudley+ at pitt.edu
Tue Dec 1 08:14:40 EST 1998


Markus Schneemann wrote:

> Hi all:
>
> 1) Hi-Fi PCR:
> For PCR you can mix Taq with a proofreading polymerase to increase
> fidelity.
> Are Vent, Pfu, Pwo equivalent?
> How much of those do you mix with Taq - 1:1 or 1:10 or even 1:100?
> Can you clone the products into TA-vectors or do you get blunt-ends from
> the proofreading enzyme?
>
> Commercially available kits (Stratagene, Invitrogen) don't tell how they
> 'blend' the taq with their proofreader.
>
> <snip>

Promega has a protocol for mixing Taq and Tfl (I think) on their web page.  I
don't know if all proofreaders are equivalent--probably not, but with a little
tinkering, you could make them all work.  Vent is very different from the
others in my non-mixing experience.  You can't T/A clone these products--the
proofreading enzymes remove the A-tail Taq leaves.

Good luck!

rich

--
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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