colony hybridize

Ned Mantei mantei at
Sun Oct 31 12:53:14 EST 2004

In article <cm31c4$mb4$1 at>,
 "BoonChoon" <boch19 at> wrote:

>I'm doing colony hybridize (E.coli) follow by Southern Blot, my colony
>hybridize seem giving me high background signal (perhaps because of the
>E.coli protein), all colony give positive signal. Anyone can help me???

If your probe was prepared by cutting a fragment out of a plasmid 
vector, you might still have some of the vector in your fragment 
preparation. If you label and hybridize this to colonies containing the 
plasmic, all will react.
This can be avoided by using PCR to prepare the probe fragment. Start 
with about 1 pg of plasmid as template, so that the amount of vector 
backbone present in your product will be negligible.

Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland

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