cosmid sequencing

[D.L. Knudson]

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
> =======================================================================

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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/
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