> -----Original Message-----
> From: Anthony Yeung [SMTP:AT_Yeung@FCCC.edu]
> Sent: Friday, June 11, 1999 5:33 AM
> To: Recipients of ABRF List
> Subject: Re: BAC sequencing
>
> Hello:
>
> 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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