HiFi PCR and RE:TritonX100

[John F Mackay]

Markus,
Pfu and Pwo are equivalent in fidelity but are higher than that of Vent. 
For ratios, you'd be best to check out Wayne Barnes' papers. With regard to 
the ends, I can only talk for the Expand (Taq and Pwo) blends but products 
have been cloned into both T-tail and blunt vectors with similar 
efficiency. However, various factors influence T-tail efficiency (primer 
sequence) and length of final extension so check archives for discussion on 
this  (PIG-tailing and a BioTechniques paper respectively).
Our PCR buffers don't contain Triton X-100 .... perhaps you are thinking of 
Promega?
Cheers,
John
Roche NZ

>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.
>2)RE:TRITON X-100
>I posted about Triton X-100 as a buffer ingredient for PCR and my feeling
>that it would decrease sensitivity about 10fold. We tested it against
>laureth12 side by side, and the sensitivity is 10x lower with Triton. Most
>likely, some peroxides in the triton harm the Taq.
>Boehringer sells their buffers with TritonX-100, but apparently their
>Triton doesn't do it...
>Cheers,
>Markus
>Markus Schneemann
>schneema at cmgm.stanford.edu <mailto:schneema at cmgm.stanford.edu>