Help for sequencing GC-rich sequence

Jim Hughes jhughes at pinnacle.jr2.ox.ac.uk
Mon Nov 30 04:01:39 EST 1998


Hi John
I take it your talking about a CpG island?  I've sequenced through a
couple of these before but with an ABI, however, I think these
approaches should work which ever chemistry you use. Addition of 10%
DMSO (final conc.) to the reaction helps decrease the melting
temperature of the DNA quite significantly and I found denaturing the
plasmid with NaoH before adding it to the reaction helped alot for the
really difficult templates.  If neither of these work you could make
some M13 single stranded preps, which I resorted to when I was really
desperate.
 If you have a friend with an ABI get them to try the new chemistry "big
dye and Taq FS" on your template as their progression through GC rich
areas has improved alot over the last couple of years and has made the
whole process much easier.

My protocol for the alkaline denaturation ca be found at
(http://worf.molbiol.ox.ac.uk/~kclark/ds_DNA_prep.html), its
specifically written for Promega wizard preps but it works from all
miniprep sources I've tried so far.

Anyway good luck

Jim

Jim Hughes
Institute of molecular medicine
Oxford



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