cdna pcr

Nicholas Plummer nick.plummer at
Mon Apr 3 08:58:27 EST 1995

In article <k9mhc.17.0003723B at>, k9mhc at (Mr Vid
Mohan-Ram) wrote:

> hi...i want to pcr up around 2kb cdna...having not much luck..anybody
have any 
> hints / tips on either cdna synthesis or the pcr conditions...?

I'm currently trying to amplify a couple of 2.5 kb cDNA fragments.  I can
make some suggestions regarding PCR conditions, but I'm still trying to
synthesize a reliable cDNA template myself.

Anyway, I've had good luck amplifying fragments up to 4 kb in length using
Boehringer Mannheim's Pwo polymerase.  I use extension times of about
three minutes.  For first strand cDNA templates, I generally do 30 rounds
of PCR, and then take 1 microliter of the product as template for 30 more
rounds with nested primers.  Often, a product will not be visible after
the first amplification but will appear after the second round.

As for the cDNA synthesis:  you might try making cDNA with primers that
will reverse transcribe your target cDNA specifically.

Hope this helps


Nicholas Plummer
Graduate serf
Department of Human Genetics
University of Michigan
nick.plummer at

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