competent bacteria

Tom Dougherty Doughert at synapse.bms.com
Fri Feb 16 09:40:12 EST 1996


In article <4fqe85$1eu8 at columba.udac.uu.se>, Anna Blom <Anna.Blom at bio.embnet.se> says:
>Hello!
>Does anyone has a good method of preparing competent bacteria that can be
>frozen?
>My bacteria are OK as long as I do not freeze them (5x107cfu/ug). If I
>freeze them they become almost useless - 105 cfu/ug. I use the standard
>calcium method. I tried to add DMSO or glycerol while freezing bacteria
>in liquid nitrogen, but it did not help.
>Any good advises?

Anna,
  When preparing competant cells for storage, treat them with CaCl2 on ice,
as per the standard protocol, and then add glycerol to 10% while they sit on
ice.  The glycerol must be added slowly, with gentle mixing, as the glycerol
releases heat when dissolving.  The trick is not to let the cells warm up.  
After the glyceol is dissolved, put aliquots of a convenient size (0.2ml) into
sterile tubes that are prechilled on ice.  Freeze rapidly in Liq. N2 and store
at -70.
                             Tom

while they sit on ice



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