direct sequencing (35S) from PCR product

[Steven Tait]

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 sequenced PCR products directly after cleaning them up with 
Wizard PCR preps, NaOH de-naturation as per plasmid sequencing in the
Sequenase manual, then following the Sequenase 
protocol except that the termination mix has 10% DMSO added 
(the slight change in termination mix conc. doesn't seem to matter).
The results could be a wee bit temperamental but might be worth 
giving it a bash, you may even be able to get away with ethanol
purification of the PCR products.
Steven Tait