PCR with 7-deaza-2'-deoxyguanosine triphosphate

galloway at dogwood.botany.uga.edu galloway at dogwood.botany.uga.edu
Tue Sep 19 12:24:27 EST 1995


I'm trying to amplify a 1.1kb gene fragment from genomic DNA and/or cDNA from 
various sources.  I have what I believe to be a pretty good set of degenerate 
primers (64-1024 degr, Tm approx. 37 degrees C) and have a plasmid with the 
cloned gene as a positive control.  At times I get great amplifications, but 
most of the time I get nothing (or so it seems recently)... even from the 
positive controls and reamplifications(?!).  I've gone through the 
usual (and time consuming) processes of trying to optimize (different TAQ, 
machines, annealing temps, concentrations of dNTP, primers, MgCl, etc.) with 
little or no luck.  Sometimes the reactions work, and sometimes they don't.  8(

Someone suggested using 7-deaza-2'-deoxyguanosine triphosphate in a 3:1 
mixture with dGTP in my dNTPs.  Has anyone tried this?  Any comments or 
suggestions?  Any help would be MOST appreciated!  8)

Thanks in advance,
  -Greg-

******
Greg Galloway
Department of Botany
University of Georgia
Athens GA  30605
galloway at dogwood.botany.uga.edu



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