Trivial transformation improvement

David Faguy 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?
-David
~~~~~~~~~~~~~~~~~~~~~~~~~~~
David Faguy
Assistant Professor
Dept. of Biology, University of New Mexico
(505) 277-3609;  fax (505) 277-0304






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