semi-specific PCR

[Dr. Andrew Bahn]

Dear netters,

I'm working on first strand cDNA-libraries that were immobilized on
magnetic beads. I try to amplify a "native" well known clone out of one of
these libraries with one specific primer (universe primer) and an oligo
dT25 (reverse primer). I tried different alternatives: 1. two rounds PCR,
then taking the supernatant for further PCR amplification; 2. 5 or 10
rounds PCR and then taking the supernatant for PCR; 3. PCR with the library
for 35 cycles; in every case a standard PCR protocol with an annealing of
52-55=B0C was used. I always get a smear!! But if I test the specific revers=
e
primer I get a big specific band! I can't understand what went wrong. What
is the problem?
Please help asap!

Best regards

Andrew =20

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Dr. Andrew Bahn
Institut of Physiology and Pathophysiology
University of Goettingen, Humboldtallee 23, D-37073 Goettingen
e-mail	: bahn at veg-physiol.med.uni-goettingen.de
phone	: +49-551-395894; FAX	: +49-551-395883