I would be interested to know the primer lengths you use for BAC clone
sequencing please.
Tony
At 01:31 PM 6/10/99 -0700, Leviten, Dina wrote:
>Hello All!
>
>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
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
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