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PCR conditions for DHPLC ?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Jan 31 11:26:33 EST 2000


In article <8742i0$khm$1 at willow.cc.kcl.ac.uk>, Colin Dolphin
<colin.dolphin at kcl.ac.uk> writes
>It appears to me
>that the "best enzyme" might be a highly processive enzyme (most standard
>Taqs) in combination with a dash of Pfu, or equivalent, to add
>proof-reading activity together with a hot-start facility achieved,
>perhaps, by adding appropriate antibodies. 

I would not call Taq highly processive! It has very poor processivity
(40-60bp) but is better by a factor of 2-4 than Pfu etc. Don't forget
that all enzymes currently used for PCR are repair enzymes not
replication enzymes.

Does Pfu need to be hot-started? Stratagene in a former Strategies
claimed that Pfu had minimal activity at RT so there shouldn't be a
problem with it. 

So try a 'Gold' Taq with Pfu or an ordinary Taq with antibody and Pfu.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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