M13 cloning problem

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Wed Jan 17 11:57:04 EST 1996


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

: We are having problems cloning a fragment into M13mp19. It's not so much
: the fragment as that we are getting pinprick sized plaques (I'm not even
: sure you could call them plaques more like a single cell etc) even with
: the original Boehringer M13mp19 RF.

First thought of the cuff...make sure the seeded cells are in log phase.

: If I infect the same cells with
: M13mp19 phage then we get normal plaques. My suspicion is that because
: we are electroporating the ligation mix we are zapping out the F'.

After zapping, are you then mixing the transfectants with log phase cells in
the overlay, or just plating the zapped cells in the overlay?

: 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?

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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