Electroporation Improvements

Ben-Sapiens srutt at beagle.Colorado.EDU
Fri Sep 9 20:33:52 EST 1994


Hello fellow zappers,

I have been trying to improve my transformation frequencies so that I can
create a large plasmid library. Here are some details and results so far:

E. coli strain: JM107 (w/ single-copy Cm plasmid)
optimal OD600 = 1.5 (yes, better than 0.5, 0.9, 1.3)
optimal kv/cm = 12.5 (couldn't try anything higher)
transformation efficiency of 50ul cells (w/ 1ug plasmid)  = 5X10^7
live cells per 50ul sample = 7.5X10^7 (30X lower than literature)
cell survival after electroporation = near 100%
fraction of surviving cells which receive plasmid = 67%
pantoyl lactone (pantolactone) doesn't seem to help

problems:

How can I increase the amount and density of live cells?
I'm obviously doing something very wrong in the preparation
and/or handling of the competent cells such that while they
are competent - they are sparse. Has anyone observed this
kind of problem before? If I could increase the number of
live cells by 30 fold then theoretically my transformation
frequency would improve by as much (1.5X10^9) and then I
would be ready to proceed with making the library.

Thank you very much for your advice,

steve ruttenberg      srutt at boulder.colorado.edu



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