At 10:50 AM 3/28/01 -0600, Sheryl Christofferson wrote:
>Greetings-
>I have a question from one of the researchers in
>my group. He would like to run fluorescent samples
>on a native sequencing gel: that is, one without
>urea. Has anyone ever tried this on any automated
>sequencer? I'm curious as to whether the analysis
>parameters would have to be different.
>
>Thanks in advance-
>
>Sheryl Christofferson
Dear Sheryl,
I read your correspondence with interest : The answer to your question is
'yes it has been tried on an automated platform' but in the context of SSCP
analysis rather than sequencing.
Exclusion of urea from the gel will render the gel non-denaturing - hence
the necessity for SSCP, where conformational states must be retained for
analysis.
Another consequence of leaving out urea would be to speed up electophoresis
considerably : I have played around with Urea conc. in 377 gels for the
purposes of making them more denaturing and it has a big effect on fragment
mobility.
Thus, I suspect the analysis parameters might be similar but the run
voltage and/or power may have to be reduced.
My advice would be to talk to AB who have practical procedures for doing
what you propose but in the context of SSCP : There techsupport number is
1-800-831-6844 x22 (for 377's)
Hope this helps a little !!
Laurence
******************************************************************************
Laurence Hall,
Einstein Genome Center,
1695 Poplar Street,
New York 10461.
Tel. (00) 1 718 405 8380
Fax. (00) 1 718 405 8383
E-Mail : LHall@megabase.aecom.yu.edu
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