protocol for JM109 competent cell
fred at here.there.com
Tue Mar 3 19:35:10 EST 1998
In article <34F6D2D0.5225 at imcb.nus.edu.sg>, mcbzzs at imcb.nus.edu.sg wrote:
> Dear friends,
> I am looking for a good protocol for making high efficiency frozen E.
> coli competent cells (JM109 strain). I have tried RbCl method, but the
> cells transformation efficiency are very poor. Could anyone send me a
> better protocol or let me share some of your good experiences. Thanks a
> lot. Jackson
> e-mail:mcbzzs at imcb.nus.edu.sg
I've used JM110...I think that JM109 is pretty much the same.
The difficulty is that JM110 is dam- and dcm-, and introduced DNA seems to
fare poorly...that is, the lack of restriction systems in this strain
makes for very poor transformation with methylated (i.e., normal) DNA.
My solution was to use electroporation- this was the only way I got
acceptable transformation efficiencies. It isn't any harder to make
e-competent cells, perhaps even easier. Just grow up cells to the usual
OD (0.3 or so, early to mid log phase), place on ice until cold, then
harvest at 0 degrees C, and wash 3x with ice cold 10% glycerol in double
distilled or better H2O, reducing the resuspension volome each wash until
at the end you've got 1/1000 the original volume. More washing may prove
helpful if arcing occurs from too many dissolved salts left from the media
(DNA must be essentially salt free as well...ppt., wash 2x w/ 70% EtOH,
and resuspend in good water). Keeping the cells ice cold through out is
essential. Aliquot into single transformation sized amounts, and for
storage at -70, flash freeze w/ EtOH-dry ice bath.
Assuming you have access to an electroporator of course.
If not, something else I've tried is to do the necessary constructions
using less difficult cells (JM101 works great...it grows really fast,
unlike DH5a or XL1-Blue), then use relatively large amounts of DNA and
cells to get JM109 with your plasmid inside...then you can prep up your
non-methylated DNA for further use.
The point is to use JM109 _only_ to make your DNA non-methylated...for
cloning steps, as you've seen, it sucks.
I hope this helps.
em-ay-kay-are-oh-ell at students.wisc.edu
(translate the first part into letters to use this address)
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