sequencing post-lineraization

Alex Laufer laufer at renovis.com
Thu Oct 11 16:08:00 EST 2001


Hi all,

Perhaps one or more of you have thoughts on the subject.  I'm attempting to
sequence internally in a BAC.  The read goes out ~200 bps into a nasty
repeat region (mostly GT, w/an occasional A) and then signal dies off,
probably due to secondary structure.  I've tried Big Dye (v. 2 & 3), dGTP,
dGTP & Big Dye 2 combined, & dRhodamine terminator chemistries, with varying
amounts of DMSO & cycling conditions.  The best result has always been with
Big Dye 3, but its still not good enough.  The latest advice from ABI was to
use a rare cutter or topoisomerase to linearize the template prior to
sequencing.  Has anyone tried either of these methods, and specifically, is
there any cleanup necessary prior to sequencing?  Is there any thing else I
should attempt (aside from shotgunning the BAC)?

Thanks in advance for your advice, Alex

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