Sequencing directly from gel slices ?

Alexander Kraev kraev at bc.biol.ethz.ch
Wed Aug 28 11:58:14 EST 1996


In article <321E3D1C.3C09 at bcc.orst.edu>, "Bryan L. Ford"
<fordb at bcc.orst.edu> wrote:

> Does anyone have a reliable method for directly cycle sequencing using
an excised
> agarose gel band as template, without further purification? My typical
template
> would be a strong PCR product in the range of 500 bp. I would use a nested
> sequencing primer. I have a vague recollection of some protocol using
LMP agarose
> dirctly in this regard, but cannot presently locate any references.
Thanks for any
> assistance you may give me.

Call FMC, they should give you the protocol and the reference for sequencing
from Sea-Plaque agarose. It is a rather obscure publication, but the idea is to
cut out a band from this low-gelling temp. agarose, melt it, dilute and 
introduce into a cycle sequencing reaction. You should have an idea which volume
of agarose contains 25-50 pmol of your fragment. That's it.

I hope this helps,



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