IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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.

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

More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net