DNA stuck in wells in gel retardation assay

gc genecutl at mendel.berkeley.edu
Sat Jan 22 16:45:20 EST 1994


In article <2hov4vINNq76 at jhunix.hcf.jhu.edu>, zxi at jhunix.hcf.jhu.edu (Xin
Zhang) wrote:

> 
> Dear netters,
> 
> I am using a 200bp DNA fragment in the gel binding assay, the DNA is
> amplified from plamid by PCR, gel purified, phenol extracted.  The DNA
> runs fine by itself in the acrylamide gel, but some batches of DNA would
> stuck in the wells if CRP binds to this DNA, other batches would not.  I
> am really confused about it.  Anyone can tell me what could happen to
> DNA to make it stuck in wells?
> 
> xin zhang
> Johns hopkins university

I think it's a good idea to wash out the wells of the gel right after
you take the comb out to get rid of unpolymerized acrylamide.  Another
thing which may help is including NP40 in your binding reaction, gel, and
buffer (I use .05%).  This helps to get rid of non-specific aggregation.

-- 
--gc
 



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