Blue/white colony selection problem.

Rafa Maldonado Rafael at genetics.med.utah.edu
Thu Sep 1 19:57:37 EST 1994


In article <Pine.3.89.9409011650.A17675-0100000 at mucc.mahidol.ac.th>
gscwnw at MUCC.MAHIDOL.AC.TH (Wises Namwat - SCMI - 3636183) writes:

> growing on amp&x-gal plate can be use for selection the success insertion 
> clone, the white colonies. Theoritically, the white colonies must be 
> bigger than the blue ones.
 

I don't think so. Why? My white colonies are the same size than the
blue ones.

> But the smaller white colonies always appear 
> around the blue colony. These smaller clones contain the different size 
> of plasmid. Does anybody know what are these smaller colonies, and where 
> does their plasmid come from?

Probably, the samll colonies are of type "satellite colonies", and they
normally appear in Amp plates around an amp(r) colony. B-lactamase is
excreted to the medium, and there it degradates ampicillin: some
neighbor cells can take adventage of this excreted enzyme, and they
grow, but slowly because they are not Amp(r) per se: they don'y have
the plasmid and they are white, then. If you can catch plasmid from
them, probably is the wrong one.
To advoid satellite colonies, don't incubate Amp plates too much (16 h.
is OK), use high concentrations of ampicillin (100-200 ug/ml) or use
carbenicillin (100 ug/ml).

I hope this helps...

Rafa


----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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