Trivial transformation improvement
dfaguy at unm.edu
Fri Feb 11 15:30:12 EST 2000
ChenHA <hzhen at freeuk.com> wrote in message
news:38A35D23.F01C9B0F at freeuk.com...
> It's the reverse. You need recovery time for Kan because it inhibit
> protein synthesis, therefore without a recovery period the gene product
> that produce Kan resistance is not made. For Amp type antibiotics, the
> cell wall synthesis is affected but cell only get lysed when the cell is
> expanding or dividing. There is therefore time for the cells to produce
> beta-lactamase before the antibiotics cause any serious damage. I
> sometimes plate the cells straight after heat shock (if I'm short of time)
> and it seems to work well. However I have not made any comparison of
> transformation efficiency with and without incubation.
In my old lab we also often skiped the recovery step. One week someone tried
three times to sub-clone and never got any transformants. He was Kan
selection and not allowing the cells to recover.
I have question: in my cynical way I've always thought companies used
longer recovery times than needed so that transformation efficiency would go
up - presumably after about 15-20 minutes the cells are dividing and your
'efficiency' is artificially higher. Does anyone know the optimal time for
uniquely transformed cells to recover?
Dept. of Biology, University of New Mexico
(505) 277-3609; fax (505) 277-0304
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