cosmid sequencing

Peter J. Myler mylerpj at u.washington.edu
Wed Jan 3 13:07:13 EST 1996

Hi folks:

We are about to embark on a large scale cosmid (genome) sequencing
venture and I would like to survey people to get their opionions on a
couple of alternative approaches.  We are planning to shotgun clone
cosmid and sequence using an ABI 373A (and hopefully a 377 later).

1.  subcloning - is it better to use M13 (ssDNA - better read lengths?)
or a ds plasmid (e.g. pBluescript - better for joining contigs, 2
sequences per template).

2.  screening subclones. Does it make sense (in terms of money & effort)
to screen subclones (by restriction digestion and hybridization) to
eliminate no-insert clones and clones containing cosmid vector DNA or
just go ahead and sequence everyting and sort it out later?  What about
E.coli contamination?

3. sequencing reaction - dye primer vs dye terminator? When should you
use TaqFS vs Thermosequenase?

4. what sort of read lengths are people routinely getting (from 373A,
with & without stretch and 377)?

Thanks to everyone in anticipation of your response.



=======================================================================Peter J. Myler                         phone: (206) 284-8846x332
Seattle Biomedical Research Institute  FAX: (206) 284-0313
4 Nickerson Street                     e-mail: MYLERPJ at U.WASHINGTON.EDU
Seattle, WA  98109-1651

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