Gene fishing

Susanne Rohrer 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
want.

Susanne





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