Gene fishing

David Reeves dcr22 at cam.ac.uk
Tue Mar 28 08:34:18 EST 2000


----------
>From: Susanne Rohrer <srohrer at immv.unizh.ch>
In article <38E0AFE3.B8EF5476 at immv.unizh.ch>, Susanne Rohrer
<srohrer at immv.unizh.ch> wrote:



>(BIG smile on face) It just worked half an hour ago (that is, over night) for
>the first time.

Congratulations!

>
>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

I'm interested that you use a proofreading enzyme - I have been using Taq
because everyone else doing this seems to and I thought that proofreading
would actually be a bad thing because of that same effect - ie it  would
give me more non-specific products (I already get quite a few). But if it
works I'll definitely try it, thanks!



David Reeves
Dept Biochemistry
University of Cambridge UK




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