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competent cells stock - (Jul/10/2011 )

we're run out out of our competent cells stock...it is due to freezer breakdown. i try to grow them back from the glycerol stock, but it seem not working anymore. if i buy a comercially competent cells from company, how do i make a glycerol stock from it?
thank you..

-pLASMID-

pLASMID on Mon Jul 11 06:38:12 2011 said:


we're run out out of our competent cells stock...it is due to freezer breakdown. i try to grow them back from the glycerol stock, but it seem not working anymore. if i buy a comercially competent cells from company, how do i make a glycerol stock from it?
thank you..


just grow the bacteria overnight (say some 5ml culture without Ab), next day morning add 500 microlitres of the grown culture into 500 microlitres of Glycerol in LB (70% Gly in LB) mix and freeze. This is how i do.. may be some others would give you even other way to make efficient glycerol stocks...good luck

-GNANA-

I usually prepare 40% glycerol in LB as stock (Stock A).
For an overnight culture in 500ul LB broth, I add equal volume of "stock A" to make a final volume 1ml with concentration of 20% glycerol stock.

-Adrian K-

You don't need to buy more competent cells, you just need some more of the strain you have been using. It is really easy to make your own: have a look under the "Eschericia coli" link on this page http://openwetware.org/wiki/Protocols.

-bob1-