Gel Shift Probe

h9290184 at hkucc.hku.hk h9290184 at hkucc.hku.hk
Tue Apr 5 01:49:36 EST 1994


In article <smrodems-010494000151 at f181-052.net.wisc.edu>, smrodems at students.wisc.edu (Steve Rodems) writes:
> 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?
> 
> 
> -- 
> Steve "Some day I will get the hell out of Wisconsin" Rodems
> 
> "Then I am here for the Lee family renioun ... shur-wajo-shur"
 Hi Steve,
Is it possible that your ds probe get denatured as you handle it?
I usually see three bands on my gel shifts, one stronger band at the bottom
which is the ds probe and two fainter bands which sometimes do not get resolved
are the two strands of the denatured probe. Freezing and thawing the probe is
sometimes good enough to denature it. You can test for this possibility by
adding one of the oligos unlabeled and see if any of the upper bands get
shifted down to the ds band.
Please post consensus.
Good Luck.

Kai Min Chan
The Institute of Molecular Biology 
The University of Hong Kong
Hong Kong
H9290184 at hkucc.hku.hk




More information about the Methods mailing list