Inoue again....

James Graham graham at biodec.wustl.edu
Sun Jun 16 13:10:09 EST 1996


You may need to use liquid nitrogen to freeze. Cells prepared by
the Inoue method are less competent if used directly without freezing
-freezing increases effieciency a log of two.

Otherswise try to obtain a cell culture grade of DMSO. Redistiled
is best from freshly opened bottle (store aliquots).

Jim
J. Graham
Biology Department
Washington University of St. Louis

doesburg at pa1.fgg.EUR.NL 
(Paul) writes:

>HI,
>I've tried making competent cells according to the Inoue protocol but 
>just can't reach a competence of 10^8, let alone 10^9, with either 
>DH5alpha or XL2BLUE cells(stratagenes ultracompetent cellstrain). I seem 
>to be stuck at around 7*10^7. My reference plasmid is from a commercial 
>source so I assumed I must be doing something else wrong. Now I remember 
>the question being asked, but couldn't find a reply whether freezing and 
>storing the cells at -80 instead of N2 decreases the competence. And 
>is LB soft agar really needed?



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