Sequencing high G + C DNA

ajeffs at chmeds.ac.nz ajeffs at chmeds.ac.nz
Sun May 7 15:45:23 EST 1995


In article <3o5dk4$rv3 at synapse.bms.com>, Steven Goldberg <goldberg at bms.com> writes:
> 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.

Our lab. uses Sequenase (or T7 Pol.) with the termination temperature 
raised to 48oC. I have successfully sequenced GC-rich templates containing
repeat sequences with only this change to the standard Sequenase protocol.
Bumping this to 50oC has also worked fine.

Cheers,
Aaron

> 
> Steve Goldberg 



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