PCR with degenerate primer

Jill Kellogg jkellogg at epitope.com
Fri Feb 6 12:49:23 EST 1998

on 2/4/98 qin ling wrote:

>I am trying to fish out the cDNA of which 20aa N-terminal seq is known. I
>have linkered cDNA as template. So in my PCR, one primer anneals to the
>linker, the other one is a set of nested degenerate primers designed based
>on aa seq. But I couldn't get any specific bands out of my PCR.
>Anybody has done similar experiments with success?  Please give me some
>briliant suggestions. 

When I fished out a gene in a similiar experiment I found that getting good cDNA
was essential, because unless the RT reaction goes all the way to the start of
the mRNA your degenerate primer won't find its homologous sequence.  I used
several RT's and found that AMV-RT from Life Sciences Inc. (St. Petersberg,
Florida) was the one that worked for me.  (No affiliation with Life Sciences.)

Good luck,

Jill Kellogg
Research Associate
Agritope, Inc.
Beaverton OR

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