sequencing single gel band

ANDREI POPOV ANDREI.POPOV at bbsrc.ac.uk
Sun Jun 18 07:07:40 EST 1995


>I'm trying to sequence a PCR band off a polyacrylamide gel,the other bands
>on the gel represent internal standard molecules which I need to separate
>from my band of interest. I have tried using costar collumns and freeze
squeezing the cut band, but when I try and PCR the isolated fragment I get
multiple bands, which is unusual for my primer set. Is this due to the
salt in the sample from the gel? If so has anyone any ideas how I can
isolate this band better.
Thanks
Gareth
 
Dear Gareth,
try to cut down the number of cycles and/or primers concentration.
BTW, before freeze squeezing you might dialyse (wash) the agarose block
in water 

regards

Andrei        




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