sequencing from polyacrylamide gels

Carol A Dimeo cdimeo at UDEL.EDU
Tue Sep 2 16:43:47 EST 1997


Does anyone have a protocol for elution of distinct bands of DNA from
polyacrylamide gels (such as DGGE gels) for subsequent sequencing (with
the FS kit from ABI)?

Here's my problem:  I've been amplifying communities of bacterial DNA with
PCR and running these samples on DGGE gels to separate the different DNA
within each PCR sample.  Each sample produces several bands of DNA that
separate beautifully, and I would eventually like to sequence each band.
When I've cut the bands out and used a little piece of the
gel (polyacrylamide)  slice in a re-amplification reaction, I get a
beautiful re-amp (as it appears on an agarose gel).  Unfortunately, when I
run this amp back out on a second DGGE gel to confirm that I have isolated
and reamplified the band of interest, I find that the PCR reaction has
reproduced ALL of the bands that were in the original PCR sample.

This phenomenon is consistent...Despite multiple tries, I have never been
able to isolate and re-amplify only one band.  Ultimately, I want to
sequence these bands, so it is very important to me to develop a protocol
I can rely on.  It seems to me that the PCR reaction is the culprit, which
is why I am trying to find a method of circumventing the re-amplification
step altogether and sequencing the DNA directly from each band in the
polyacrylamide gel. 

Has anyone else encountered this problem?  Does anyone have a protocol
that works?

Thanks!

Carol A. Di Meo
cdimeo at copland.udel.edu
Graduate College of Marine Studies
University of Delaware 




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