How to get a plasmid into WT E.coli
Doughert at synapse.bms.com
Fri Mar 15 09:17:13 EST 1996
In article <4h9r4g$rjn at info.usuhs.mil>, jim rogers <rogers at usuhs.usuhs.mil> says:
>Need some help.
>We are trying to get a plasmid into a clinical isolate of E coli.
>Transformation does not work, so we have tried electroporation,
>conjugation using different methods. We can get pBR328 in but not
>a RK6 based plasmid. The control using DH5 work. My first thought
>was that maybe it had to do with the oriR of the second plasmid
Have you considered that it might be a restriction/modification problem?
It's conceivable that the clinical isolate might have an active system that
is degrading the plasmid, particularly if it is large. The DH5 strains are
restriction minus, and this may allow the second plasmid to establish
itself. The other possibility is plasmid incompatibility. Have you checked
the clinical isolate to see if it carries a natural plasmid? If it is an R6K
based replicon, then your incoming plasmid would not be able to
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