Sequencing Primers for Automated Sequencing

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Dec 24 10:02:00 EST 1992


In article <1992Dec22.220623.2753 at samba.oit.unc.edu>, plc at med.unc.edu (Philip L. Carl) writes...
>From: plc at med.unc.edu (Philip L. Carl)
>Newsgroups: bionet.molbio.methds-regnts
>Subject: Sequencing Primers for Dye Terminators
>References: 
>Sender: 
>Followup-To: 
>Distribution: world
>Organization: UNC-CH School of Medicine
>Keywords: 
>I am posting this on behalf of a colleague who runs the automated
>sequencing facility here.  She would like to obtain the sequence of
>primers that have worked for priming vectors other than the M13 forward
>and reverse primers.  She uses the ABI Taq sequencing system and would
>particularly like to know about experiences with primers such as T3, T7,
>SK, KS (for Bluscript vectors)  and SP6, and the lambda gt11 forward and 
>reverse primers.  Please include any information about sequencing
>conditions if they differ from the standard ABI protocols.  I believe
>there is a new newgroup for automated sequencing questions, but I've lost
>the address, so if someone could supply it and/or forward this notice to
>that newsgroup I would appreciate it.  Thanks
> 
>Phil Carl
>Program in Molecular Biology
>and Biotechnology, UNC

Hi,
	Question specifically pertaining to automated DNA sequencing
can be addressed to:

	autoseqs at net.bio.net

As for your somewhat of an answer to your question regarding different
primers you listed, the problem is complex.

First, if you are using the Taq fluorescent terminators, then just pick
primers using the usual criteria for stringency, uniqueness, etc that
you would use for radiolabeled sequencing.  This has been discussed in
detail in this forum before and no need to address it here.

Second,  if you plan on making the dye-labeled primers, then you are
in big trouble because you will need software, which is NOT supplied
by ABI on a routine basis, to create a "mobility file" for each specific
primer sequence.  ABI has done this for many of the more popular primers
and these files either already are there in the ABI folder in your system
folder on the Mac.  For primers other than these, at one time ABI was
willing to create this file for the few sites that wanted to use different
primers but the last time I tried to get them to do this for us nothing 
happened for 3 months so I gave up.

Third,  although many of the primers on your list already have mobility files
already made, I'd suggest you contact ABI directly and see if they have any
advice.

Cheers.........bruce

Merry Christmas!                                              .
         Chappy Channuka!                                    .|,
                  Divine Divali!                             -*-
                           Creative Kwanzaa!                 '|`
                                    Joyous Solstice!          :
                                             Happy Holiday!
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -  - - -



More information about the Methods mailing list