Help! my colony blots are lighting up like a christmas tree

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Feb 16 11:36:17 EST 2000


"R. Jayakumar" wrote:

> Hi
>    Somebody should really help me out with this problem.  I did colony lifts
> of about 1000 clones onto Both nitrocellulose and Nylon (N+) colony lift
> membrane discs and did a normal hybridisation with my DNA probe labelled
> with p32 using the GIBCO BRL's random primer labelling kit.  But nearly all
> my colonies lighted up, with both the membranes I used.
>     Can anybody tell me where the mistake is?  For your information, I
> followed the colony lift technique given in Maniatis manual.  As
> recommended, I have also given prewashes of the blot before doing
> hybridization.
>     Any suggestion will be most welcome
> thanking you in advance
>
> cheers
> jayakumar

Hi Jayakumar,
Sounds like your probe hybridizes to the bacterial genome or a region in a
plasmid. The simplest guess would be that the probe you labelled was
contaminated with plasmid DNA that now lights up all bacteria that have the
plasmid...

Hope this helps,
Frank





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