sequencing high GC DNA

Richard Hopkins hopkins at numbat.murdoch.edu.au
Thu May 4 21:59:20 EST 1995


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

> 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.


I have found that the Promega fmol sequencing kit has been great for
sequencing my 85% GC rich template.  Initally, I tried sequencing direct
from PCR product and while this worked OK I found that I was able to get
much cleaner reads once I cloned the fragment into a T-vector.  I also
find that adding DMSO (5%) to the cycle sequencing reaction helps to
produce a more accurate read.  I think I can remember reading somewhere
in Biotechniques that adding DMSO in a sequenase type reaction can also
help to sequence GC rich templates.

I hope this helps

Regards

Richard Hopkins

Veterinary School
Murdoch University
Western Australia



More information about the Methods mailing list