direct sequencing (35S) from PCR product

Roz McDonnell rmcdonn at macollamh.ucd.ie
Tue Sep 24 07:34:16 EST 1996


In article <51ocuv$rnt at whitbeck.ncl.ac.uk>, "D.D. Morgan"
<D.D.Morgan at ncl.ac.uk> wrote:

> Jeremy Marcus (marcusj at HELIX.MGH.HARVARD.EDU) 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
> 
We are currently using a method of sequencing from pcr products that
employs the sequenase kit.  The method was posted in 1 Mar 1995 by C.P
Palmer and we have found it to work extremely well.  pcr product is cleaned
using a Geneclean kit (we use Qiaquick pcr purification kit) and eluted
into 30ul of TE.  Template mix is as follows:
6ul cleaned pcr product, 1ul primer (@ sequenase recommended concentration)
2ul sequenase 5x buffer and 1ul DMSO.  The mix is boiled for 5 min and snap
cooled on ice. Follow the sequenase protocol after denaturation and
annealing from here.  The following modifications are made: Termination
mixtures are made up containing 10% DMSO and the termination reactions are
performed at 50 degrees celcius.  I hope this helps.  You may wish to look
at the original message.

Rozalyn


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