Hi all,
We have several protocols related to this on the protocol page at:
http://www.genome.ou.edu/proto.html
Of immediate interest might be the information in:
"Large Scale Plasmid, Cosmid, BAC, PAC, and Fosmid DNA Isolation by
a cleared Lysate Method Followed by Double Acetate Precipitation"
http://www.genome.ou.edu/DblAcetateProcV3.html
"Sequencing off Cosmid, BAC, PAC, and Fosmid Templates with ABI Big Dye
Terminators" http://www.genome.ou.edu/big_dyes_cos_bac_pac_fos.html
Enjoy......bruce
***************************************************************************
Bruce A. Roe, Ph.D George Lynn Cross Research Professor
Department of Chemistry and Biochemistry
University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at ou.edu
********************** http://www.genome.ou.edu/ **************************
You write:
=>
=> >I am looking for information on sequencing bac's with regard to the new
=> >'big dye' chemistry of the ABI Prism 377. How do you prepare your
=> >template, cycle sequence reaction, etc. I have been unsuccessful in my
=> >attempts with stardard ABI protocols with regard to template and primer
=> >concentrations.
=>
=> Dear Mairead,
=>
=> we have used the following:
=>
=> about 12 microl volume,
=> 8 microl premix
=> 10 pmol primer
=> 3 microg DNA
=> - 4' 98 C
=> - 30 cycles
=>
=> In principal, it worked. Our users have used Qiagen colums for template
=> purification. Still, some people get better DNA than others. As usual (or
=> here even more), template quality is crucial.
=>
=> Best regards, Bernd
=>
=> PS: In case that you get more personal replies, could you post a summary =
=> to
=> the net? BW
=>
=> _____________________________________________________________________
=> PD Dr. Bernd Weisshaar
=> MPI f=FCr Z=FCchtungsforschung
=> Carl-von-Linn=E9-Weg 10
=> 50829 K=F6ln
=> Germany
=>
=> Tel. office: +49-221-5062 590 Tel. lab: +49-221-5062 311
=> Fax: +49-221-5062 313
=> e-mail: weisshaa at mpiz-koeln.mpg.de
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=>
=>