competent cells stock - (Jul/11/2011 )

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

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

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.

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.