PCR sequencing with the ALF (Question)

Bruce Roe BROE at AARDVARK.UCS.UOKNOR.EDU
Tue May 12 07:49:00 EST 1992


MIKA SALMINEN <MSALMINEN at nphi.fi> writes:

Subject: Re: Fluorescent sequencing from PCR templates
To: BROE at aardvark.ucs.uoknor.edu

=>
=> Hello! We have just bought a Pharmacia ALF sequencer. Could you possibly
=> share your PCR-product sequencing products with us? Thanks in advance,
=>
=>              Mika Salminen
=>              msalminen at finnphi

Hi,
        I have a couple of thoughts for you.
1. Contact Pharmacia.  You just spent >$100,000 US for an instrument
   and they should give you a protocol.
2. We use Taq Cycle sequencing on the ABI 373a essentially following
   the protocol described by ABI in their kit (although we do not buy
   their kit but instead make our own).  We've modified the protocol
   as described in:

S.L. Chissoe, Y.F. Wang, S.W. Clifton, N. Ma, H.J. Sun, J.S. Lobsinger, S.M.
Kenton, J. D. White and B. A. Roe. Strategies for Rapid and Accurate DNA
Sequencing. Methods: A Companion to Methods In Enzymology. 3, 55-65 (1991).

3. The problems with sequencing off PCR templates are:
        a. purity of the template
        b. amount of the template
   There is more art than science in template isolation and I'd suggest
   just trying gel elution by your favorate technique and various template
   DNA concentrations in your standard sequencing reactions.  Also I'd
   advise starting with something simple, such as ss-m13, get that to work
   and then move on to ds-pUC and finally going to PCR products after you
   get the ds-vectors to sequence well.

Cheers and good luck,
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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