Ampicillin-Resistant! - (Jan/18/2005 )

recently, our working ampicillin does not work in cloning. even in negative control, the dh5alpha e.coli cells grow well. instead of adding ampicillin into LB during pouring the LB plates, we have tried to add ampicillin together with the bacteria just before spreading plates. Here are some questions I would like to ask:

1. how long can be ampicillin (dissovled in distilled water) stored at -20C and (in powder) at RT?

2. also, after dissolving the ampicillin powder into distilled water, the solution is passed through a 0.22uM filter. is it suitable to use a 0.22uM filter or we should use a 0.45uM filter?

3. is it necessary to use autoclaved distilled water or NANO water to dissolve the power? does this affect the results?

Thank you very much.

Dear

Ampicillin sodium (Wako) can be solved in milliQ or autoclaved water after vortexing about 1 minute. To keep it in sterile condition, we always use 0.22 mikrometer filter in clean bench. To increase the solubility, you can use 30% of ethanol.

How hot your media when you added ampicillin. It should around less than 60 centrigre celcius.
Hopefully, this can help you

I always keep ampicillin (powder) in 4 degre (referigerator). Solved ampicillin can be storage in -20 degree for long time (1 years).

yes, there is a possibility of inactivating the ampicillin by high temp. however, we have tried to add the ampicillin to the bacteria in LB just before spreading the bacteria on plates. so this shouldnt be the reason.

do you know how pure your dh5-alpha bugs are? Maybe one or two plasmid carrying cells managed to sneak their way into your vial.

Try a new tube of competent cells. Try a new batch of ampicillin as well.

Or borrow cells/ampicillin from a neighboring lab to test.

Ampicillin does't actually kill the cells -- it merely prevents the formation of a cell wall and thus prevents cell division. When you mix ampicillin with bacteria before plating, all that happens is the bacteria are chilling out waiting for that ampicillin to diffuse into the agar, thus decreasing the concentration to the point where even non-resistant cell can grow.
Try using a 55C temperature bath: After taking the agar media out of the autoclave let it rest in the 55C bath for at least an hour. This temp is hot enough to prevent gelling but cool enough not to destroy the ampicillin.