Separating ssDNA from dsDNA (asymmetric PCR) ???

Francisco G da Nobrega ib - bio 7588 fgdnobre at usp.br
Wed Oct 11 09:46:11 EST 1995


On Tue, 10 Oct 1995, bmay wrote:

> Does anybody out there know a simple way to separate ssDNA from dsDNA=20
> (~250bp) and other compounds in the PCR mixture after asymmetric PCR ?=20
> I want pure ssDNA for sequencing without expensive and time consuming=20
> procedures as gel-cutting and gel-extraction-kits and so on.
>=20
> Thanx a lot in advance
>=20
> Bernhard Mayr
> Medizinische Hochschule Hannover
> Dept. Clin. Endocrinology
> Hannover, Germany
>=20
>=20
Dear Bernhard:
I guess that you can run your sample in a polyacrylamide gel (5-6%) in=20
1/2 TBE at room temperature or in the cold room. This is the standard=20
procedure used routinelly for the few people  that still use the=20
Maxam-Gilbert sequencing strategy (for separating dsDNA from the single=20
strands and the single strands thenselves) Today people use it for the=20
SSCP technique looking for mutations. The dsDNA usually runs fast and the=
=20
single strand lags behind.
=09=09
=09=09=09=09F.G. Nobrega
=09=09=09=09Biologia - IBUSP
=09=09=09=09Universidade de S=E3o Paulo=20




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