IUBio

Sequening bacs with big dye chemistry?

Bruce Roe BROE at aardvark.ucs.ou.edu
Mon May 4 11:54:31 EST 1998


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
=> WWW-URL: http://www.mpiz-koeln.mpg.de/~weisshaa/BW-info.html
=> 
=> 





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