jim rogers <rogers at usuhs.usuhs.mil> writes:
>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
Lessee...been a while since I addressed this kind of thing.
If the plasmid has been propagated in other strains, then it must
have an intact origin of replication. You have solid markers on
it, like antibiotic resistance, right?
The quickest explaination that comes to mind is that it is vulnerable
to the target strain's restriction endonucleases. Is your plasmid
methylated at the same sites as the two which were successfully
Also: is the target cured of all plasmids? Some plasmids are not
compatible with others.
Other than these two, all I can come up with is stuff you have likely
already thought of.
Hope it helps.
Dept. Biochemistry & Microbiology