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What's The Problem With Ampicillin Selection?

by on 9th of May, 2011 in Cloning & Expression

ampicillin.pngAmpicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it serves it's purpose, there can be problems using this selection marker if the user is unaware of it's limitations. This article provides a quick overview of what these limitations are and how to avoid them.

The basis of ampicillin selection is the hydrolysis and inactivation of the antibiotic by beta-lactamase expressed from the plasmid-borne bla gene. Here's the problem: beta-lactamase is secreted by the bacteria. The resulting build-up of extracellular beta-lactamase can inactivate the ampicillin in the culture medium, removing the selective pressure, if the culture is not handled properly.

In liquid cultures, this means that a portion (possibly a very large portion) of the cells no longer have the plasmid, giving poor yielding plasmid preps, protein expression etc. On agar plates, ampicillin degradation can lead to the formation of satellite colonies on transformation plates. Satellite colonies are very small colonies of cells that have not taken up the plasmid that form around a large colony that has taken up the bla-containing plasmid. The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony. The satellites are not necessarily a problem as they will not grow when transferred to a medium containing fresh ampicillin.

So how do you avoid plasmid loss when using ampicillin as a selection marker? Here's how:

  • Don't allow liquid cultures to saturate for too long. I would recommend never growing cultures higher than OD600=3 (in LB)
  • If you are using a starter culture, always pellet and re-suspend the starter culture in fresh, antibiotic-free medium before innoculating the main culture. This is to remove the secreted beta-lactamase from the medium.
  • Use a higher ampicillin concentration if you are experiencing problems. I would recommend using 200 micrograms per mL or higher. This makes it harder for the beta-lactamase to inactivate all of the ampicillin and is especially useful for avoiding satellite formation.
  • For the same reason, never use old ampicillin stocks or plates as the ampicillin will have broken down somewhat, giving a reduced effective ampicillin concentration.
  • If all else fails, switch to carbenicillin selection. This antibiotic is also inactivated by beta-lactamase but more slowly than ampicillin is. For this reason carbenicillin selection is far more effective than ampicillin. Unfortunately it is much more expensive!

Do you have any tips on working with ampicillin? If so, please leave a comment.

About the author: Nick Oswald
I started Bitesize Bio on a Macbook on my kitchen table in 2007 while in my 7th year of working as a molecular biologist in biotech. My aim was to share the know-how that I had acquired from the school of hard-knocks in the lab, so that others could learn from my mistakes and small victories. Nowadays my mission is to facilitate the gathering of hardcore know-how from whole spectrum of bioscientists and share it here on Bitesize Bio to create a super-mentor that any bioscientist can turn to for much-needed guidance.

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4 thoughts on “What's The Problem With Ampicillin Selection?”

  1. Avatar of DrKennB DrKennB says:

    I have never had a problem with ampicillin in liquid cultures. Yes, on plates you can get a decrease in ampicillin concentrations such that you get satellite colony growth but I do not think that you can ever deplete the concentration in liquid to the point where non-resistant colonies will grow.

    Kenn Buckley

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  3. Avatar of MugeAtis MugeAtis says:

    I have an question.Can the bad smell of ampcillin stock(mixed with EtOH and water)be the sign of degredation of antibiotic?After a certain time such as 3 weeks,my 100ug/ul amp. stock starts to smell.Should I take it serious?

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