gel retardation assay

Song Tan tan at aeolus.vmsmail.ethz.ch
Sun Sep 4 10:25:05 EST 1994


In article <34ceeh$bec at mserv1.dl.ac.uk>, "shantaram bharadwaj"
<sb at biotech.ssf.ernet.in> wrote:

>         Could someone help me out with solving a problem I encounter
>         during my gel retardation runs. I seem to get a large blob
>         towards the lower half of my gel.
[snip]

>         I do not purify my probe and does it cause any trouble (esp with
>         Klenow sitting around)?. The blob is observed even in the absence 
>         of the protein (labelled DNA with Binding Buffer)

My guess is that the large blob you observe is unincorporated radioactive
nucleotides.  Presumably this large blob migrates faster than the free DNA
band.  

You can remove unincorporated nucleotides by passing the probe through a
Sephadex G25 Medium spun column in TE (Maniatis et al describes how to
make and use spun columns).  I usually phenol/chloroform extract my DNA
probes after labelling but before passing through the spun column to get
rid of any enzyme or other proteins (BSA) in the labelling mix.

Hope this helps.

-- 
Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email:  tan at aeolus.vmsmail.ethz.ch



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