direct sequencing (35S) from PCR product

Pamela Norton pnorton at lac.jci.tju.edu
Wed Sep 18 13:08:38 EST 1996


In article <323F0655.48E at helix.mgh.harvard.edu>,
marcusj at HELIX.MGH.HARVARD.EDU (Jeremy Marcus) wrote:

> Is direct sequencing of pcr products a possibility using 35S manual 
> sequencing?  The Sequenase protocol seems to keep popping up as a 
> useful tool for direct sequencing, but most people seem to be using an 
> automated system or 32P.  Ideally, I'd like to find a way to 35S 
> sequence directly from a pcr product of ~400bp.  Am I dreaming?	
> Yvonne Barnes
> barnes at helix.mgh.harvard.edu

   It can be done. The biggest problem that I had was getting enough PCR
product to serve as template in the reaction. In the end, I cut out the
bands from the first amplification (we were characterizing alternatively
spliced products), reamplified each individually duplicate or triplicate,
then pooled the sets of reactions and used a Centricon filter unit to
remove primers and dNTPs. The results were not great, but adequate to
determine the site of the palice junction, as all the sequence was known. 

   Good luck,

      Pam Norton

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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