Non-radioactive sequencing method ??

Richard V. Giles giles at liv.ac.uk
Fri Oct 16 03:13:47 EST 1998


Alternatively, use a standard PCR sequencing protocol (such as the Promega
Fmol kit), substitute 5' fluos or 5'dig labelled primers, diffusion blot the
fragments from the PAG to nylon membrane, and detect using an alk-phos /
anti (fluos/dig) antibody and NBT/BCIP.

Simple.
Cheap.
Read length >250bases (starts ~5bases from the end of the primer).

This is how I do it regularly.

Richard

Szatmari George wrote:

> <delete at leeds.ac.uk (Hedley Carr) () wrote:
> : Hi all,
> : I'm looking for a non-radioactive DNA sequencing method that fulfils
> : the following criteria:
> :
> : -Rapid to perform
> : -Cheap
> : -Base reading only need be 100-150 bp per lane.
> :
> : OK, I'm dreaming, but I'm going to be sequencing rather a lot of short
> : sequences so massive reads aren't necessary. I would also prefer
> : non-radioactive methods because:
> :
> : a) I value my gonads
> : b) Radioactive materials are a pain to handle and are useless after a
> : month of storage.
> :
> : I've checked out a Cycle-sequencing method with silver-staining but
> : that didn't work particuarly well last time (poor signal to noise
> : ratio).
> :
> : Any ideas?
> :
> : Thanks,
> :       Hedley Carr.
> :
>
> Hedley,
>
> You have several options:
>
> 1-Promega's Silver Sequence Kit (basically silver stains your seq gel)
> 2-Boehringer Mannheim's Dig-sequencing kit (bit pricey)
> 3-find a place that has an automated sequencer
>
> George
>
> : P.S. To reply via e-mail, remove the 'XXX' from my address.

--
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
http://www.liv.ac.uk/~giles/





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