HiFi PCR and RE:TritonX100

John F Mackay MackayJF at bmnz.co.nz
Mon Nov 30 20:59:58 EST 1998


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>




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