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..
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#2
GNANA
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Posted 11 July 2011 - 12:34 AM
pLASMID, on 10 July 2011 - 10:38 PM, 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..
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
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#3
Adrian K
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Posted 11 July 2011 - 09:39 AM
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.
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.
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#4
bob1
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Posted 11 July 2011 - 03:52 PM
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.
