DNase FOOTPRINTING Off a Gel-Retarded Complex

Ken Doyle kdoyle at promega.com
Thu Dec 1 13:40:04 EST 1994


In article <3bde8u$l6p at bigfoot.wustl.edu>, ramu at artsci.wustl.edu (Thiruvamoor P. Ramkumar) says:
>
>
>Hi,
>        i'm looking for help on doing DNase Footprinting from a 
>DNA-Protein complex isolated from a gel-retarded complex. Primarily, my 
>concern is to elute the DNA-Protein complex out of the gel without 
>disrupting the complex. Possible buffer compositions, elution conditions, 
>quantitation parameters etc. will all help. 
>        
>
> _
>ramu
>
>
It would be diffcult to do this, depending on the dissociation rate
of the complex.  What is easier to do is to footprint within the gel slice,
then elute the DNA fragments and run your denaturing gel.  This is best 
achieved with chemical reagents such as Fe-EDTA or copper-phenanthroline,
however it can be done with DNAseI, except that it takes about 10X as much
enzyme as is needed for solution footprinting. The gel slice is resuspended
in a standard DNAse footprinting buffer (containing Ca/Mg) and is treated
with DNAse I: the amount and time would need to be optimized depending on
the volume of your gel slice.


Ken



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