Gel Shift Probe

Martin Leach leach at mbcrr.harvard.edu
Fri Apr 1 10:41:18 EST 1994


In article <smrodems-010494000151 at f181-052.net.wisc.edu>,
smrodems at students.wisc.edu (Steve Rodems) wrote:

> Iv'e been seeing some strange things with 32P labeled probes in my gel
> shift experiments.  In a control lane with no extract added my probe is
> consistently two bands about 1 cm apart at the bottom of a 5-10%
> polyacrylamide gel (all other lanes have the same thing).  Sometimes I get
> a couple other bands above and below the major ones.  I fill in 5'
> overhangs (4 nt) to label, my label is the first nucleotide added in the
> fill in, and it labels only once on each end.  The DNA I use has been
> either gel purified 30 bp or two 30 nt oligos annealed.  I thought that
> maybe it could be incomplete fill in so that I have a range of labeled
> probes different by 1-4 bps but I wasn't sure if the separation would be
> that great.  Does anyone have any ideas or has anyone seen this before?


I believe this happens if the DNA can form tertiary structure...

eg. Form triple helixes.

I'm not sure about this, but it may account for extra complexes seen
without any protein - and when gel-purified

Flame me if im talking out of my gilson!!

Martin
-- 

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