PCR enzymes and Non-radioactive DNA sequencing

Jerry Kropp jkropp at dnai.com
Fri Apr 25 22:48:09 EST 1997


In article <5jjehv$bl6$1 at cnn.ksu.ksu.edu>, met at gizmit.wheat.ksu.edu
(michael Tilley) wrote:

> Experiences with non-radioactive DNA sequencing, particularly Promega Silver 
> stain kit. Although the Amersham system looks nice I do not have the ability 
> for large transfers.
> 
> Regards
> MIKE

Mike,
I've been doing non-R/A sequencing for a couple yr now, but I use a
bio-primer for detection via the Sequenase protocol. That notwithstanding,
the transfer is a breeze:
Leave your gel stuck to one plate after the e'phoresis
Lay a water-wetted piece of uncharged nylon on it
Put one dry sheet of Whatman 3MM or the equivalent on top of that, and the
other plate on it
Allow it to sit 15min., zip-zop, you transferred it
UV X-link if you want to impress the boss or let air-dry O/N--makes no
difference
I detect with a kit from NEBL, Photope, I think they call it.
Works fine, and v. sensitive, but bands are not as sharp (read publication
quality) as with 35S.
HTH,
Jerry



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