long PCR

Tom Knight via methods%40net.bio.net (by tk from mit.edu)
Fri May 4 23:07:15 EST 2007

"Mandeep Sharma" <mxs781 from psu.edu> writes:
> I am trying to amplify a 10kb maize genomic DNA fragment using long PCR.
> I have already tried Pfu ultra fusion from promega and geneamp high
> fidelity PCR system from applied biosystems and they didn't work. If you
> have tried amplification of this size fragment and you got the
> amplification then can you please suggest which taq polymerase should I
> use and what will be the most suitable conditions for long PCR with that
> taq?

I've had good luck with the Roche Expand long template PCR kit.  Use a
maximum of 68C extension temperature.  If you have low GC, use 64C.
If you have high GC, add 5% Betaine.

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