Competent cells at OD = 0.94

David Micklem dmicklem at cmgm.nospam.invalid
Wed Mar 13 11:57:37 EST 2002


In article <3C8F6AC2.962D86E at genome.wi.mit.edu>, Peter Ianakiev
<ianakiev at genome.wi.mit.edu> wrote:

>Hi guys,
>Has someone tried to make competent cells at OD600=0.94?
>Original paper has been published by Tang, X. et al. 1994 Nucleic acids
>Res. 22, 2857-2858.  I wonder if someone reproduced the results.
>Any suggestions of making ultra competent (library grade) cells will be
>appreciated. Inoue method looks good, but has an  insufficient yield for
>our needs.
>Thank you for your help
>Peter Ianakiev

Wow, you must be doing some seriously high-throughput stuff if you
can't get enough cells from the Inoue method: I find a single batch
lasts me for months. Or maybe I've misunderstood, since growing to
OD600=0.94 wouldn't give sooo many more cells that the (very flexible)
OD's suggested in Inoue et al anyway.

One suggestion is to use fewer cells/transformation. For moderately
high throughput expts I've done transformations with 20ul Inoue cells
in 200ul PCR tubes with good results.

And you could always just grow up 10 (100, 1000, however many) flasks
of cells to give you what you need....


Ah, oh, maybe you mean the Inoue method doesn't give you
competent-enough cells? I don't know of any method (for chemical
competent cells) that works as well or as reliably as the Inoue one,
although I'd be happy to hear otherwise. Batches do vary a bit, so
trying again might help, otherwise you might need to use
electroporation, which generally gives the highest efficiencies.

HTH,

David

-- 
D.R. Micklem,              Time flies like an arrow... 
Dept. of Genetics,          Fruit flies like a banana.
Cambridge University,      Email:dmicklem at cmgm.stanford.edu 
Cambridge, UK              Phone: +44 (1223) 766336
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