cdna pcr

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


In article <k9mhc.17.0003723B at rfhsm.ac.uk>, k9mhc at rfhsm.ac.uk (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

Nick

------------------------------
Nicholas Plummer
Graduate serf
Department of Human Genetics
University of Michigan
nick.plummer at med.umich.edu



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