FLUO SEQUENC

Bruce Roe BROE at AARDVARK.UCS.UOKNOR.EDU
Sun Dec 22 07:39:00 EST 1991


Jean Francois Chasse writes: 
=> =========================
=> 
=> We are looking the users for a PCR sequencing protocol
=>  for automated sequencing analysis
=> A.L.F. DNA sequencer transfert ( Pharmacia ).
=> 
=> Any protocol of users is welcomed ( included cycle sequencing ).
=> 
=> Dr CHASSE Jean Francois
=> URA1335, labo de biochimie genetique
=> Hopital NECKER
=> 75743 PARIS cedex 15 - FRANCE
=> mail : EARN::"CHASSE at FRCITI51.bitnet"
=> FAX  :  (331) 42 73 06 59   france
=>                                                    Thanks
=> 
=>                                    Dr CHASSE Jean Francois
=>                                    M.D., Ph.D.
=>                                    Associate professor
=> 
Bonjour,
        I have added your name to the Fluorescent Sequencer User List
I have been compiling and will send the list to you as a separate message
after this one.
        Regarding sequencing of PCR products on the ALF.  Since we
do not have an ALF (we have 2 ABI 373A's) I cannot give you any exact
help.  I believe Rick Wilson and the group at the MRC-Cambridge, England
use their ALF's for just this and there is nothing they do differently
with their sequencing.  The "tricks" seem to be in PCR template purity.
There are several methods to purify PCR products from low-melt agarose
gels, such as Glassmilk, direct phenol extraction, etc. but the best
seems to be to make the eventual template strand using Biotin-labeled
and then purify the template using Streptavidin/magnetic beads.
        Uhlen's group in Sweden and Lloyd Mitchell at the NIH have
published methods for incorporating and purifying biotin-labeled PCR
products. I think in Nucleic Acids Research and PNAS, respectively but
do not have the references handy. The Ansorge Group at EMBL in Heidelberg
also may have a protocol for this.
        Good luck and let me know what you find so I can post for all.

                                Cheers.............bruce
  **************************************************************************
 *  Bruce A. Roe                    Professor of Chemistry and Biochemistry *
 *  Dept. of Chem. and Biochem.     INTERNET: BROE at aardvark.ucs.uoknor.edu  *
 *  University of Oklahoma          BITNET:   BROE at uokucsvx                 *
 *  620 Parrington Oval, Rm 208     AT&TNET:  405-325-4912 or 405-325-7610  *
 *  Norman, Oklahoma 73019          FAXnet:   405-325-6111                  *
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