ADVICE ON SSCP WANTED

M K Bennett pamkb at mail.bris.ac.uk
Fri Mar 11 06:07:57 EST 1994


MED6MHS at VAX.BIO.LEEDS.AC.UK wrote:
:     HI,
:       I would be grateful if anybody would offer me any advice with 
:       regard to setting up SSCP to look for a single base change in
:       a PCR product of 204 b.p,
:                              Thanks in advance,
:                                                Max.

I did try this with limited success with a 100bp PCR product which had a
single base pair deletion.  I used the method of looking for heteroduplex
formation rather than SSCP.  I also used constant temperature with a
gradient of denaturant rather than constant denaturant and a gradient of
temperature.  Basically I did this because I hadnt access to a temperature
gradient apparatus.  It is relativly straight forward the only problem I
had was keeping the temperature high enough when loading the gel.  When I
neared the end of the project i did order some of the hydrolink mutation
detection enhancement electrophoresis gel which looks as though it is a big
improvement over using normal acrylamide, I have no idea what they have
done to it to improve it. They also say that if you use this gel that
you dont need gradients or the temperature gradient apparatus.  They
can also supply a heteroduplex control DNA which was what I could have
really done with when I was setting up the technique.  There is a chapter
in PCR protocols: a guide to methods and applications by Innis, Gelfland
etc which deals with SSCP.  I also have some original refs which I can give
you if you like.

Mike
______________________________________________________________________________

Dr. Mike Bennett D.Phil
Dept. Pathology and Microbiolgy		E.Mail M.K.Bennett at Bristol.ac.uk
University of Bristol.			Tel No. 0272-288597
					Fax.No. 0272-303501



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