Differential Display PCRs - help needed!

Miss J.M. Cameron jcameron at hgmp.mrc.ac.uk
Wed Mar 27 13:24:20 EST 1996


I am currently having terrible trouble remamplifying cDNAs that I have cut out from
differential display gels.

My method is to cut out the band from the gel, elute it for an hour in TE and then
boil it for 5 minutes.
   I usually try PCRing 1, 2 and 3 microlitres of the eluted DNA in a 20 microlitre
PCR reaction (2.5uM anchor primer, 0.5uM 10mer primer, 20uM dNTPs, Promega Taq and 
Promega buffer with Mg2+). 

   Sometimes these PCRs work, and sometimes they don't. At the moment I can't get 
them to work at all!!!

   Has anyone had success with this stage of the DDRT process??

Thanks for your help!

Jessie Cameron
Galton Laboratory
Univesity College London, UK








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