double-stranded sequencing GC-rich inserts
USA::RGB12955
RGB12955%USA.decnet at USAV01.GLAXO.COM
Thu Jul 16 10:52:00 EST 1992
Netters:
We've been trying to double-stranded sequence Bluescript inserts from
a cDNA that is about 68 - 70% G+C (Sequenase version 2.0 kit,
using Stratagene's -20 and reverse primers) with extremely mixed
results. Mostly what we've been seeing is either no sequence at all or
darkly smeared lanes, depending on the insert and the primer. We
suspect that the annealing conditions are to blame, as the DNA is
clean (Magic-mini) and the primers are new, plus we've gotten it to
work okay for one template. USB said that linearized DNA would
make any secondary structure/annealing problems worse, and
suggested doing the extensions on ice and the termination reactions at
50C. It didn't solve the problem, and only gave very faint sequences.
Does anyone have any suggestions?
Rich Buckholz/Mary Martinson
Glaxo Research Institute
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