M13 cloning problem

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Thu Jan 18 11:39:26 EST 1996


Dr. Duncan Clark (duncan at genesys.demon.co.uk) wrote:

| Electroporation is very efficient at curing plasmids but I would have
| expected the F' to stay in.

> Is this your own observation? Could you please give more details or a
> reference?

: One of mol.biologists at a previous job used to cure plasmids regularly
: this way. Swears by it. We find consistently that if we are zapping a
: plasmid into a strain that already contains a compatable plasmid we get
: much better duel selection if we add in more of the compatable plasmid
: as well. 

Could this be a survivor problem, ie. those that survive are transformed
with both plasmids? Maybe this kind of stress causes them to actively kick
out (?) the resident plasmid. I've heard people say this kind of thing, but
I don't think there is any evidence for an active involvement in the ousting.

: The M13 has sorted itself by just using chemically competent cells. I'll
: pass on why electropopration gave us a problem.

Duncan, this is really interesting though. Can I guess? Maybe the electric
shock doesn't cure F, but instead destroys much if the F pili receptors (?)
and lowers infectivity. It would be fun to see what proportion of cells have a
selectable F marker (not Tn10) after zapping with no DNA added.

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