High Efficiency Transformation (libraries)
tan at aeolus.vmsmail.ethz.ch
Sun Feb 20 11:02:08 EST 1994
In article <CLDvws.EAC at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu
(the End) writes:
>Thanks to Mike Coady and Rafa Maldonado for pointing out the Inoue
>In growing cells at 18C prior to Ca treatment, has anyone arrived at a good
>inoculation and growth period ? I hear a couple "overnight" references,
>but I would prefer not to determine the doubling time at this temperature.
>I'm going at 20-22C, room temp here.
It's hard to give any clear cut recommendation for how long to grow the cells
at 18C before the Ca washes since there are so many different strains with
different doubling times out there. And then there's the question of the size
of the colonies you inoculate.
Having got those caveats out of the way, here's my procedure for preparing
competent TG1 cells by the Inoue et al method (Gene, 96, 23-28, 1991):
inoculate 10 - 12 colonies (0.5 to 1 mm in diameter) of TG1 from a freshly
streaked plate into 250 ml of media (I use 2xTY with additional 10 mM MgCl2, 10
mM MgSO4, 2.5 mM KCl) in a 2 liter flask. Grow shaking (~200 rpm) at 18C for
12 to 18 hours until optical density at 600 nm is about 0.6.
I do grow the cells overnight, but I usually inoculate one flask of 250 ml
media in the afternoon and another flask of 250 ml media in the evening. This
way, I'm pretty much guaranteed having one flask reaching the appropriate OD
sometime the next day. Yes, you do waste 250 ml media this way, but it's
better than finding that your cells have overgrown or don't reach the right OD
till late at night.
Hope this helps.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch
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