When performing RNAi by feeding assays, I understand that L4440 E. coli must be grown O/N in presence of ampicillin. But when making NGM plates, ampicillin is replaced with carbenecillin. Is there a reason carbenecillin is used in this case? Can I just use ampicillin instead?
In molecular biology, carbenicillin may be preferred as a selecting agent (see Plasmid stabilisation technology) because its breakdown results in byproducts with a lower toxicity than analogous antibiotics like ampicillin. Carbenicillin is more stable than ampicillin and results in fewer satellite colonies on selection plates. However, in most situations this is not a significant problem so ampicillin is sometimes used due to its lower cost.
Basically, for most purposes in the lab they’re interchangeable.
Thanks for your thorough reply! That clears up exactly why carbenecillin is used for most laboratory purposes, but does anybody know if ampacillin will work with RNAi? I don’t have carbenecillin and would like to use amp instead.
As Hillel explained, Ampicillin and Carbenicillin are essentially interchangable, it’s just that you stay on the safe side of town when you use the latter for your selections. Why?
Well, as Hillel also already outlined, Carbenicillin is more stable (more slowly inactivated by beta-lactamase) than Ampicillin. So, old Amp plates are a no-no, the chances of satellite colonies are increased with Amp etc. etc.
If you really don’t want to spend the extra on Carbenicillin (street price is 1g for €360 vs. ~€50 for 5g Amp) then you should:
Always use fresh Amp plates (that goes for Carb plates too).
Avoid saturated cultures where you are selecting with Amp.
Pellet and resuspend your starter cultures in fresh medium before innoculating.
Remember, 1g Carb gives you a shed load of agar…40 litres?