Summary of DNA-Protein aggregation in gel binding assay

Xin Zhang zxi at jhunix.hcf.jhu.edu
Thu Jan 27 20:43:49 EST 1994


Hi,

One week ago I posted a question about possible DNA contamination, even
though I still have not solved the problem, I summerize all the response
I got, and sincerely thank everyone who reponded.

My original question:

> 
> 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

------------------------------------------------------------------
From: genecutl at mendel.berkeley.edu (gc)

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.

-----------------------------------------------------------------

From:	"M.E. Couchman" <cou at leicester.ac.uk>

Do you ethanol precipitate your probe first? Often this problem is due to
unincorporated label.

The other cause is large protrin aggregates that stick in the wells. Try
spinning the protein as hard as you can just before using it. Apart from that,
there's notmuch you can do. Changing the binding buffer, running buffer and
competitor DNA coul all (possibly) make a difference.

M. Couchman
Leicester University
--------------------------------------------------------------
From:	Eva Lorenz <elorenz at umaxc.weeg.uiowa.edu>

Hi
Sorry, I can't help you, but I had the same probelem and we never
figured out how to solve it. I just went back to make my NDA via the old
plasmid prep procedure. We did not just have the problem of it being
stuck in gel shifts, but when we would label the DNA it would remain 
stuck in the wells too when we tried to isolate the fragment. I did not
just happen me, but to other people in the lab too who were using PCR
generated fragments for either labeling or gel shifts. I posted an
inquiry in this newsgroup earlier and got no answer. 
Sorry, I can't be of any help. If you have any more luck in getting
responses, please let me know what people suggested. 
E-mail address: Eva-Lorenz at uiowa.edu
Thanks. And good luck 
Eva Lorenz





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