Gel Shift Assay--Probe in Wells

[Steve Rodems]

In article <325p0v$h5p at usenet.INS.CWRU.Edu>, pxb26 at po.CWRU.Edu (Peter
Brunovskis) wrote:

> 
> What are the some common causes for probe not leaving the wells in gel shift
> assays?
> What are some suggestions for avoiding this problem?

One obvious suggestion is to be sure to use a nonspecific DNA like poly
(dI-dC) or poly (dA-dT).

I have found that the choice of probe makes a lot of difference.  In the
past I have tried probes that contained "extra" sequences derived from the
Bluescript plasmid.  The multiple cloning site sequences seemed to cause a
lot of problems when I used crude nuclear extracts.  If my desired DNA
sequences also contained Bluescript MCS sequences all the probe stuck in
the well.  No amount of poly (dI-dC) could get that probe out of the well
either.  However, the identical desired sequence without the Bluescript
sequences worked much better.  

There have also been times when I have gotten a little bit of probe stuck
in the wells with no correlation to anything!  Sometimes I think this just
happens if the moon isn't properly aligned with Venus. :-)

--
Steve "Some day I will get the hell out of Wisconsin" Rodems

"Then I am here for the Lee family renioun ... shur-wajo-shur"