Freezing competent bugs...

Michael Coady 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!

Hi David,
	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.

Mike

-- 
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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