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Molecular Cloning: A Laboratory Manual, Vol 1.
#Anderson-Lancet-1965 pmid=14238093
#Datta-BiochemJ-1966 pmid=5328167
#Evans-JBacteriol-1968 pmid=4971890
#MolecularCloning isbn=0-87969-577-3
[[Category:Material]] [[Category:Antibiotic]]
[[Category:Material]] [[Category:Antibiotic]]

Revision as of 22:51, 20 December 2007


Mode of Action

Inhibits the formation of cross-links in the peptidoglycan layer (which provides rigidity to the cell wall). Most effective against cells in log phase growth (since this is when new cross-links are being formed), and has little effect on cells in stationary phase.

Mechanism of Resistance

Expression of β-lactamase neutralizes ampicillin. The resistance gene is named bla or ampR. When this enzyme is expressed on a high-copy number plasmid there is significant diffusion into the extracellular medium. As a result non-resistant satellite colonies may form around larger resistant colonies.

Satellite colonies on an Ampicillin plate
Satellite colonies on an Ampicillin plate

Stock Solution

Typical concentrations of ampicillin are 50 ug/ml for low copy plasmids and 100 ug/ml for high copy plasmids. Stock solutions are typically at 100 mg/ml, so that 1 ml of antibiotic can be added to 1 liter of broth or agar. Stock solutions made in 50% alcohol remain liquid at -20 C and are easy to pipet. Cool agar to 55C or below prior to adding antibiotic.

Ampicillin available from Sigma A-9518 (Ampicillin sodium salt), FW 371.39. To make 100ml of 100 mg/ml stock solution, dissolve 10 g of ampicillin in 50 ml of water and 50 ml of 100% ethanol.


Culture plates with ampicillin can be stored at 2-8 °C for up to two weeks. Stock solutions may be stored at 2-8 °C for up to 3 weeks. For long term storage (4-6 months), stock solutions should be stored at -20 °C. At 37 ° C in culture, ampicillin is stable up to 3 days. Sigma reference

Usage Notes

A 1990 paper by Bill Studier discusses how the secreted β-lactamase can quickly consume all the ampicillin in a culture (even at 20μg/ml Amp). A stationary culture of ampicillin resistant cells can have such a concentration of β-lactamase that even a 1/200 to 1/1000 dilution will still contain enough β-lactamase to consume all the fresh ampicillin before all the non-resistant cells from the stationary phase culture have been killed. The authors recommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.

  • Barry Canton & Matt Gethers have seen this occur when using the BioBrick plasmid, pSB1A3 expressing a high level of mCherry.

Carbenicillin is much more resistant and would be preferred except for cost. Mixtures of ampicillin and carbenicillin are often used.


  1. ANDERSON ES and DATTA N. . pmid:14238093. PubMed HubMed [Anderson-Lancet-1965]
  2. Datta N and Richmond MH. . pmid:5328167. PubMed HubMed [Datta-BiochemJ-1966]
  3. Evans J, Galindo E, Olarte J, and Falkow S. . pmid:4971890. PubMed HubMed [Evans-JBacteriol-1968]
  4. isbn:0-87969-577-3. [MolecularCloning]
All Medline abstracts: PubMed HubMed
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