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E.coli not growing in liquid LB, but grows well on Agar plates - (Nov/21/2005 )

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Okay it worked now. I got nice looking colonies on my plates, they grew in mini-preps, and I just got back some sequencing results which confirmed the presence of my insert.

I changed thwo things:

1) new plates
2) I used BL21 rather than DH5alpha

Our tec thinks the plates were the problem and that the colonies I was getting initially were not actually amp resistant.


I don't think your post was particularly helpful in this case (though it is true). His problem is that his bacteria won't grow in LB+amp. If the amp was detoriated then he should get a lot of bacteria growing without the selection pressure applied (and not neccessarily his transformed bacteria only).

QUOTE (Daniel Tillett @ Dec 29 2005, 11:23 PM)
Did any of you read my post above?




When you pick the colony from agar plate and inoculate it in liquid media, where did you incubate it, in a plastic tube or in a flask?

the reason I ask this is because recently me and everyone in my lab were having a similar problem.
The difference was that if we would do a mini, 1 colony in 2 ml LB+amp, bacteria would grow.
BUT 1 ml of that ON grown culture diluted in 100-150 ml media to do a maxi, wouldn't grow at all. This happened constantly during 2 weeks.

FINALLY I figured out the problem.
The cyndosan product that we were using to kill bacteria before autocalving left over bacteria cultures, was not being washed properly even after 1 hour wash in the washing machine, therefore the residues of cyndosan in the glas Erlenmeyer flask would inhibit the growth of bacteria.

From then on. We don't put cyndosan in the culture flasks anymore. We discard the bacteria culture in a big flask named "WASTE", there we add cyndosan and that one autoclave. The culture flask we just autoclave it empty, and after we wash it and autoclave it again.


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