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



PS  Our Info Sys department forces us to put this stupid disclaimer on all our
messages.  I'm sorry they are so humorless.
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