aiyar at ebv.oncology.wisc.edu
Fri Dec 20 11:02:56 EST 1996
On Fri, 20 Dec 1996 11:03:26 -0800, LABO DE BIOCHIMIE <lbmc at sciences.univ-metz.fr> wrote:
>I am planing to use E.coli strain NM522 transformed with the pCI-neo
>plasmid (Promega). Could anybody help me in giving the procedure
>(neomycin concentration, transformation...) for selecting transformants
>on neomycin-containing medium plates? Does this strategy require special
>care to any step?
As far as I know, there is no E. coli promoter immediately 5' to the
neomycin phosphotransferase (npt) coding sequences in pCI-neo. There
is a beta-lactamase gene on pCI-neo so you can select for the
transformed bacteria with ampicillin or a similar beta-lactam.
If I am mistaken, and the npt gene can be transcribed in E. coli, you
can select for transformed bacteria in LB-kanamycin plates with
the concentration of kanamycin between 15 and 30 micrograms/ml.
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
University of Wisconsin-Madison
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