srohrer at immv.unizh.ch
Tue Mar 28 08:13:21 EST 2000
David Reeves wrote:
> Has anyone here ever had any luck using degenerate PCR to do cross-species
> cloning? I'm unsure whether to use some sort of touchdown/step up PCR
> protocol with the degerate primers or just decrease the annealing
> temperature in the standard protocol.
(BIG smile on face) It just worked half an hour ago (that is, over night) for
the first time.
I amplified a sequence from four species of staphylococci. I used degenerate
primers (22mers plus restriction sites - upper primer 96x degenerate, lower
24x). I did a touchdown for 5 cycles. before I have tried Taq polymerase but
that didn't work - someone told me Expand or just Pwo works, because it will
degrade and repair the 3' end of the primer if it doesn't fit perfectly. I
also added 1% DMSO because I got Primer dimers. I still get them, so maybe
what really made it work was just the Pwo polymerase.
I'll clone and sequence it and tell you whether it's the right thing if you
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