Ultracompetent E. coli cells?

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Fri Dec 15 12:49:37 EST 1995


Andy Phillips (andy.phillips at bbsrc.ac.uk) wrote:

| Could some kind person post or email me a quick recipe for the Inoue
| method for producing ultra-competent E.coli?

In a nutshell,

1.  Inoculate from an overnight grown in LB
2.  Grow in 250 ml "SOB" at 18C until OD600 = 0.6
2.  On ice for 10 minutes.
3.  Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
4.  Resuspend cells gently in 80 ml of ice cold "TB".
5.  On ice for 10 minutes.
6.  Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
7.  Resuspend cells gently in 20 ml of ice cold "TB"
8.  Add DMSO to a final concentration of 7%.
9.  On ice for 10 minutes.
10. Aliquot into 1-2 ml and freeze in liquid nitrogen.
11. Store in liquid nitrogen.

-----------------------
"TB":
10 mM Pipes
55 mM MnCl2
15 mM CaCl2
250 mM KCl
pH 6.7
-----------------------
-----------------------
"SOB":
2% (w/v) bacto tryptone
0.5%  (w/v) yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
pH 6.7 - 7.0 
-----------------------

@article{Inoue1990,
author = "H. Inoue
     and H. Nojima
     and H. Okayama",
title = "High efficiency transformation of
{{\em Escherichia coli}} with plasmids",
journal = "Gene",
volume = "96",
pages = "23-28",
comment = "chemical transformation of bacteria",
year = "1990"}

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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