Loss of plasmid in liquid culture

Rafa Maldonado Rafael at genetics.med.utah.edu
Wed Mar 16 20:50:56 EST 1994


     The ampicillin can be a problem, as Anne says. How much ampiciliin
do you use? For a multicopy vector as yours, you should use 100 to 200
ug/ml. Usually, this overcome the problem of finding the plasmid. The
protein product of the gene cloned can be toxic, but if you have cells
without plasmid (and without beta-lactamase gene) in a culture with
ampicillin, you should use a higher amp concetration: your are losing
the plasmid because the b-lactamase is excreted to the medium and is
inactivating all your ampicillin. 
     There are other vectors with kanamacyn resistance gene which can
be used if you still hace problems with high amp concentrations.
     Other question is the toxicity of proteins expresed in coli. If
you don't mind the protein, cloning in the reverse orientation is a
good idea, because you don't want to purify the protein. In case you
want, there are some vectors with induced expresion, inverting
promoters, repressed expression, and so. Normally, when the product is
toxic, you lose the plasmid or get reorganizations in it, when using a
strong selection. But that is another story.

Good luck!



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



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