Freezing competent bugs...

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Thu May 12 11:35:24 EST 1994


Greetings one and all...

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!

David
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+  David L. Haviland, Ph.D.	     Internet:"haviland at kids.wustl.edu"   +
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