I too would appreciate hearing from you regarding the responses that you
receive to your questions.
Preter J. Myler wrote:
>> 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.
>> Regards
>> Peter
>> --
> =======================================================================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
> =======================================================================
--
=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=
Dr. Dennis L. Knudson, Professor of Entomology and Microbiology
Department of Entomology Telephone: 970 491-7255
College of Agricultural Sciences Fax: 970 491-0564
Colorado State University Internet:dknudson at lamar.colostate.edu
Fort Collins, CO 80523 USA URL http://klab.agsci.colostate.edu/
=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=
