High Efficiency Transformation (library)

William J. Buikema wjb1 at kimbark.uchicago.edu
Tue Mar 1 13:23:43 EST 1994


In article <CLssJo.Jq0 at usenet.ucs.indiana.edu> jgraham at bronze.ucs.indiana.edu (the End) writes:
>
[stuff deleted]
>
>I prepared my first batch of room temperature Inoue cells (22C for 24 h from
>an inocculum of 0.07 OD) and got 5X10(7) transformants/ ug of CsCl prep
>plasmid. These were frozen in nitrogen and stored for four days at -70
>in a freezer. Strain INVaF', a DH1(5) derivative.
>
>I'm very pleased with this result as I have seldom (never) broken the 10(7) 
>barrier with painstakenly prepared unfrozen Hanahan or Ca/Rb cells. The 
>slightly lowed efficincey (compared to Mike's) may be due to a higher 
>growth temperature (22C vs 18C). Has anyone tried lower than 18C ?
>Inoue and coauthors claimed that lower temperatures were "impracticle".
>I'm not sure why 48 h would be worse than 24 for potential better 
>library representation.
>
How about the possibility of growing the cells at a higher temp initially,
then diluting and growing for a shorter time at 18C?  Surely it is not
necessary to grow the cells for 18-24 hours at 18C to get them at maximal
competency, is it?  (Sorry if this is covered in the paper, I have to get
to the library yet.)

Bill


-- 
William J. Buikema                       internet: hetman at uchicago.edu
Dept. Mol. Genet. & Cell Biol.            /  / --- --- /|/|  /|  /| /
Univ. of Chicago, 920 E. 58th St.        /--/ /-   /  / / | /-| / |/
Chicago, IL 60637 USA  (312)702-1081    /  / /__  /  /    |/  |/  /



More information about the Methods mailing list