Bacterial Electrotransformation/Antibiotics

Rick L lm11l at gpu.srv.ualberta.ca
Sun Aug 9 23:12:07 EST 1998


Koichi Kunitake <kunitake at salk.edu> wrote:
:    I have always read that after electroporation of bacteria, say with a
: plasmid with Amp resistance, you should allow the bacteria to recover for
: ~1 hour at 37C in SOC media before plating out. I decided to test this,
: and I electrotransformed my pBluescript into XLI-Blue, added 1000uL of
: SOC, mixed, and immediately plated out 200uL on LB/Amp/IPTG/Xgal. I took
: the remaining 800uL, and let it shake at 37C for 45 minutes, and then
: plated out another 200uL onto LB/Amp/IPTG/Xgal. Both plates had almost
: exactly the same numbers of blue/white colonies. It seems like it is not
: necessary to allow bacteria to "recover" before plating out on Amp plates.

:    Maybe the amp kills only growing bacteria, and perhaps the one
: bacterium that had the amp resistance made the B-lactamase while it was on
: the LB-amp plate, so there was no need to incubate at 37C beforehand. 
:    Do people routinely incubate at 37 for an hour before plating out? it
: seems like it isn't necessary, at least for ampicillin, and I would be
: curious to know if people have tried it with other antibiotics. 

: Koichi Kunitake
: <kunitake at salk.edu>

Sometimes I do, sometimes I don't, both work fine to me, but I never
compared them in a single transformation. In my experiment, the selection
marker is AmpR.

Rick 



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