Sequencing high G + C DNA

Steven Goldberg goldberg at bms.com
Tue May 2 08:55:16 EST 1995


I have been attempting to sequence DNA of high G + C content (ca. 72%)
that has been cloned into various E. coli plasmids.  My main problems
(couldn't you guess?) have been weak signals and stops as well as
phantom bands across all of the lanes in some instances.
The manner in which the DNA has been prepared doesn't seem to make
much difference, as I get the same results using CsCl-purified, Qiagen-
purified, or mini-preped plasmid.  I started off using the basic 
protocol given with the Sequenace kit + deazaGTP and 35S-dCTP.  I 
have also tried the Quick Denature kit with the same parameters.  
Primers to plasmid or cloned DNA were made but didn't make much 
difference.  Someone suggested including 1 ul DMSO in my reactions,
which did seem to help the stops a little (but could it also knock
down the activity of the polymerase?).  I raised the termination
reaction temperature to 42oC.  All of these changes really didn't
help a lot.  How about using 32P instead 35S?   

If anyone with a similar experience has any ideas, I and our radiation
safety department would be grateful.  Thanks.

Steve Goldberg 



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