What protocols are out there that describe: the preparation of the BAC DNA,
clean-up of DNA (if necessary), and the PCR reaction set-up? We could use
any advice!
We have been sequencing directly from the BAC clone and have been getting
variable results. Either it works or it doesn't. Out of about different 50
primers, 12 have given good sequence. I just ran 10 primers on the 3700,
and 1 gave excellent sequence (700bp), 1 gave poor sequence (50bp) and the
others did not work. (This is about the same success rate as the 373S.)
Just so you know, we have been using 1-2ug of DNA, ~12pmols of primer and
adding extra enzyme. We have tried two different PCR protocols and the one
at least gives us these hit-and-miss results. We are thinking resorting to
shotgun cloning it and just sequencing the whole thing. Let me know what
you think...
Thanks in advance!
Dina Leviten
ICOS, Corp.
Bothell, WA