Preparing competent E. coli

the End jgraham at bronze.ucs.indiana.edu
Mon Jul 11 11:14:11 EST 1994


Sorry for the redundant post.

> I have a very hard time believing such high competencies. 

I agree. No basic Ca++ or Hanhan based procedure has ever given me over 
10(6) cfu/ ug in any strain.

However, the Inoue method which has been discussed here frequently does deliver
1 X 10(8) as promised. The basis of this one is to grow the E. coli at room temp or below, and to use only Pipes, Ca, and Mn. Other additives were found to be 
inhibitory. Also a freeze in liquid nitrogen in 7% DMSO brings about the last 5X
increase.

Jim
J. E. Graham 
Biology and Chemistry Departments 


* Inoue et al. 1990. Gene 96 p.23-28


PS. Growing for one day at 37 yields better efficiencies as well, when 
lower efficiencies are acceptable. Be sure to catch the culture at 0.45 OD.




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