competent cells again!!

z z-suldan at ski.mskcc.nospam.org
Thu Jun 4 08:44:32 EST 1998


In article <l03130300b19b60eeb7ca@[134.174.248.196]>,
jaugliera at RICS.BWH.HARVARD.EDU ("John Augliera") 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 of the cells.
> 
> 
> 
> Thanks
> 
> John Augliera
> Technician
> Brigham and Women's Hospital
> Boston,MA 02115
> 
> email: jaugliera at rics.bwh.harvard.edu



We routinely add 10% glycerol (fisher brand cat#: BP210???) and we don't
autoclave it (although I have done this) nor do we filter sterilize it
although YMMV in terms of contamination.

Just one word about the glycerol. I find I need to add it relatively
slowly with a fair amount of intermittent mixing of the cells (by gentle
swirling of the cells in the 500ml centrifuge bottle) to get it into
solution. Only after the glycerol has been added to the cells and mixed
into solution do I then aliquot the cells to microfuge tubes which then go
directly on dryice/-80.

And one word about the flash freezing. Several years ago, I tested this.
To my surprise, if the cells sit on wet ice, the competency increases up
to 10 fold at 24hours, returns to baseline @ 48hrs and drops further after
that. So... sometimes, I will let the cells sit on ice in the CaCl2 up to
24 hours and then add the glycerol and freeze.

Good luck,
Zal



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