IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

zen of PCR

Gerri Newfry gnew at orion.it.luc.edu
Wed Jul 13 14:19:36 EST 1994


I have been happily plodding along, learning PCR and being pleasantly 
surprised that things seem to keep going well for me.  Well, of course, 
that had to end, and it has.  I've been trying to use first dig, and then 
32-P in my rxn.  And things completely stopped working.  I have been 
always using a positive control (my template will be a plasmid with the 
desired gene in it, to which I've designed my primers)  and the positive 
control no longer works.  First I thought it must be the dig, so I moved 
to 32-P.  Then I realized the positive control wasn't working either, so 
I thought our enzymes had gone bad.  PCR with and without enzymes (taq from 
Promega) gave the same results.  So I bought some taq from BRL.  This 
didn't work either.  So I tried the PCR without 32-P (presumably this rxn 
would be the same as what I had successfully been doing before I started 
the dig/32-P).  But hot and cold rxns gave the same results (ie., no 
bands).  Am I doomed to starting from scratch (making all new buffers, 
reoptimizing my reaction for Mg and pH, new enzymes, new lab....)????

tia...gerri
gnew at orion.it.luc.edu




More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net