roland.hubner at ua.ac.be
Tue Aug 14 11:44:28 EST 2001
> I am confident about my Taq
If that means that the amplifications work with Taq pol, then you have
probably no primer problem or template & rxn troubles... but have to
find the appropriate conditions for your HiFi-PCR with Pfu...
I would now add Pfu pol AFTER the initial denat. step of 5 min in your
plasmid PCR, use 1 ng plasmid and about 20 cycles, and INCREASE the Tm
by 5 and 10?C...
Else, spiking in a tiny amount of Pfu into your Taq PCR and keeping
cycle number low (e.g., 10 cycles for 10 ng plasmid) might give you
sufficient assurance to obtain correct sequence... [Will have to anyway
sequence check, if that is crucial]
Hope that helps,
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