bernard at elsie.nci.nih.gov
Fri Dec 20 15:21:50 EST 1996
In article <slrn5ble5g.jbo.aiyar at ebv.oncology.wisc.edu>,
aiyar at ebv.oncology.wisc.edu says...
>On Fri, 20 Dec 1996 11:03:26 -0800, LABO DE BIOCHIMIE
<lbmc at sciences.univ-metz.fr> wrote:
Yes, I can confirm that transformation of bacteria with pCI-neo
does not confer kan resistance so I assume any prokaryotic promoter
has been removed. This is in contrast to eg. pSV2neo which confers
kan resistance in bacteria and G418 resistance in eukaryotic cells.
(Stratagene's pBK-CMV does the same).
I hope that this helps.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
>>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.
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