ELECTROPORATION of E. coli
nash at nrcbsa.bio.nrc.ca
Mon Jan 24 16:45:09 EST 1994
In article <1994Jan24.195438.21221 at emba.uvm.edu>,
Brian Foley <brianf at med.uvm.edu> wrote:
>Seth Findley (findley at u.washington.edu) wrote:
>: Pardon this probable FAQ, but...
>: Would anybody have any tips for optimizing electroporation of E. coli?
>: Is 1x10e10 efficiency commonly obtained?
> Not in my hands. I have a hard time getting better than 1.0 x 10e9
>transformants per microgram of pBluescript. I use DH5alpha and XL1-Blue
>E. coli strains.
>: Does the growth medium make a difference?
> I found the most difference in the O.D. of the culture, and keeping
>it on ice throughout the washes. A low O.D. and thus early log phase
>growth was important. Also cooling the cells on ice before spinning them out
>of media and into the low ionic strength washes.
>: We are using both DH5a and DH10b.
I was curious as to why I could only get 1 X 10e9 some days and 1 X
10e10 on others with the same batch of cells.
Then I wrote down my controls on a grid... They went something like
this (from memory).
Host: DH5 alpha or DH5alpha F' - it didn't matter which.
pUC8 1 x 10e10
pUC18 1 x 10e10
pUC18 derivative (from a colleague) 1 x 10e10
pUC118 1 x 10e9
pT7/T3 alpha 19 (BRL's phagemid) 1 x 10e9
Now look at that... a ss phage origin (fd or M13) drops the
electroporation efficiency down a log.
A colleague (the one who lent me the pUC18 deriv) was able to
reproduce these results...
[ Call it the "Nash effect" after me ;-) To the humour impaired: My
tongue is planted firmly in my cheek! ]
John Nash (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences, National Research Council of Canada,
Yet another Aussie-in-exile ;-)
*** Disclaimer: All opinions are mine, not NRC's! ***
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