Abbarent migration in agarose gels: interesting problem
jakami at ucdavis.edu
Fri Jul 25 10:28:30 EST 1997
un691cs at genius.embnet.dkfz-heidelberg.de wrote:
> Hello, I have an interesting problem.
> I PCR a plasmid with one 3' primer, and several about 10
> 5' primers:
> ----> <----
> the 3' primer is always the same.
> As I know the promoter sequence in this construct, I
> can accuredly predict the size of the expected PCR products (150-1500bp).
> I get single bands in each lanes, the PCR worked well, as far as i
> can see, products were checked by southern hybridization.
> Now what happens is that several bands do not run at the position
> where I would expect them to migrate. This on a 2% gel. Then, If I run
> the samples on a 1% gel, the positions of the bands are altered again,
> but again not always to the expected position. for instance: a 450 bp signal
> migrates as 800 on a 1% gel ! I can exclude a mix-up of samples.
> Is my DNA 'bend' or what ? Where in the literature can I get additional
> info on this ?
> Could you answer by e-mail as well ?
> thanks, Clemens
> Uni Heidelberg
What you might be getting is heteroduplex formation between your
products. That is, some of the smaller fragments are hybridizing to the
larger fragments resulting in a strech of duplex DNA with a single
stranded tail. Because the tail has a much greater freedom of movement
than the "stiffer" double-stranded section, it will not migrate through
the gel matrix as easily and move more slowly the a fully
double-stranded fragment of equal size. I have seen this same sort of
thing with and internal 15 bp loop on a 250 bp fragment. The apperent
size (in acrylamide) was 1600 bp! Try useing single pairs of primers as
a control and size standard.
Blue Rose Biotech
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