gel retardation assay

shantaram bharadwaj sb at
Sun Sep 4 07:26:57 EST 1994

Hi Netters,

        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. I'm in the process of trouble 
        shooting but any comments are welcomed.
        I run a 5% acrylamide(80:1) gel pH 7.6 after a pre run of 1 and
        a half hours at 150 to 200 volts. In the process of troubleshooting,  
        I have eliminated the following:
       - pH (both gel and running buffer at 7.6)
       - BSA,dI-dC
        Is it possible that glycerol(10%) or/and the various other
        components as HEPES,KCl,MgCl2 and EDTA can cause such a blob?
        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)

        I would like to hear from anyone who's encountered this problem
        ASAP. Please mail me at the address below

        Thanks in advance
Shantaram Bharadwaj
sb at

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