sequencing from polyacrylamide gels
gbe392 at merle.acns.nwu.edu
Wed Sep 3 10:54:16 EST 1997
Carol A Dimeo (cdimeo at UDEL.EDU) wrote:
> 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.
Hi Carol -
I have occasionally had the same problem, although to a lesser extent. I've
been sequencing DNA out of DGGE gels for a while now, and it usually works
fine. Sometimes the band I cut out is a heteroduplex, and will give me
three (2 homoduplexes and the heteroduplex again) bands on a DGGE gel when
I re-amp, or I find that two bands have co-migrated
(I can usually straighten this out by changing the gradient on the gel).
Also, if the re-amp gives me one bright band and one or two very faint bands
on the DGGE I'll use it for sequencing anyways and most times get clean
sequence. I have to admit I don't always bother to re-run bands on the DGGE
gels once I'm familiar with a particular sample.
How many bands do you usually see from a sample? Do you see a lot of
heteroduplexes? (They tend to look different from 'real' bands).
Hope this helps, e-mail me if you want more details-
g-berardesco at nwu.edu
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