competent cells again!!

Chen Ho An chen at bsm.bioc.ucl.ac.uk
Thu Jun 4 08:56:06 EST 1998


John Augliera (jaugliera at RICS.BWH.HARVARD.EDU) wrote:
: 	I recently posted a message asking for a protocol and storage
: conditions for making bacterial cells competent.   Someone suggested
: the procedure in "Maniatis".  I read the protocol which uses successive
: washes of cold CaCl2 and the final step calls for flash-freezing the cells in
: cold CaCl2 and placing at -70C.  It makes no mention of adding a
: cryoprotectant such as glycerol.  Would anyone know if this is OK!! or
: would the addition
: 10% glycerol adversely affect the competency or viability 

I think you should add glycerol.  However I normally used 7-8% DMSO which
I find much more convenient (no need to sterilise).  Just add DMSO then
snap-freeze in liquid N2.  If it is stored in liquid N2 the cells could
last for a very long time.




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