Gene fishing

R. John Lye rjl6n at
Tue Mar 28 07:03:20 EST 2000

David Reeves wrote:
> Has anyone here ever had any luck using degenerate PCR to do cross-species
> cloning? 

Sure, I've done it quite a few times.

> 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.

You'll probably need to try both - sometimes one protocol will work,
and sometimes the other will.  In this sort of semi-non-specific PCR,
you just need to play around until it works.  I'd try a simple PCR
with a slightly reduced annealing temperature first, and if that
doesn't work then try the touchdown protocol.
John Lye
rjl6n at

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