direct sequencing (35S) from PCR product

Tom Duncan duncant at cross.bmb.hscsyr.edu
Wed Sep 25 12:41:02 EST 1996


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


I have used the Sequenase protocol for sequencing PCR products with
consistent success. As long as your PCR yields a specific fragment, the
treatment with ExoI + SAP (shrimp alkaline phosphatase) gets rid of PCR
primers and dNTPs, so you can sequence directly without having to purify
the PCR product first. The other main point is to have a relatively high
yield of PCR product to use as template; I usually start with a fairly
large amount of template (100-400 ng plasmid miniprep) and use PfuI
polymerase for amplification. Amersham sells a kit for this that
includes the ExoI & SAP but, if you already have a regular sequenase
kit, you can order ExoI & SAP separately. Good luck,
 
	Thomas M. Duncan
	Dept. Biochemistry & Molecular Biology
	SUNY Health Science Center
	750 E Adams St,	Syracuse, NY 13210
	Email:	duncant at vax.cs.hscsyr.edu or
		duncant at cross.bmb.hscsyr.edu



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