Freezing competent bugs...
coady at ERE.UMontreal.CA
Fri May 13 07:36:20 EST 1994
In article <2qtlscINNknf at medicine.wustl.edu> HAVILAND at KIDS.WUSTL.EDU (David L. Haviland, Ph.D.) writes:
>Normally, our lab has used the usual 50% (25% final) glycerol for freezing
>bacteria. Having recently found the Inoue et al Gene paper via Jim Graham,
>I've been experimenting with freezing competent bugs (XL-1 mrf', and DH5-a)
>in LB with 7% DMSO. Does anyone have any tips or suggestions using DMSO?
>I centrifuged the bugs (both of them) and then resuspended them in LB
>containing 7% DMSO (two different bugs done on different days). I had
>successful transformations with the DH5-a's but not the XL-1's. With the
>XL-1's, my plates were simply blank. I knew something wasn't quite right
>when after the hour at 37'C in LB (SOB) the bacterial cell density hadn't
>increased. I figured the bugs were dead.
>Does anyone have any experience in DMSO freezing of bugs? Specifically
>what I'm interested in is 'how tolerant is this system'? Given pipetting
>errors, will 6% or 8% work? Or is it death by freezing if I happen to use
>6.5% and not 7%?
>Any thoughts? Thanks in advance!
I've used the procedure many times with Xl-1 mrf' and it's
always worked fine for me. I've added 7% as they suggested; sorry,
but I've not tried other concentrations, though I doubt that this is
responsible for what you're seeing.
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.
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