Ampicillin

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The authors reccommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.
The authors reccommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.
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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.
 
==References==
==References==
Molecular Cloning: A Laboratory Manual, Vol 1.
Molecular Cloning: A Laboratory Manual, Vol 1.

Revision as of 19:58, 20 November 2005

Contents

Mode of Action

Inhibits the formation of cross-links in the peptidoglycan layer (which prvides rigidity to the cell wall). Most effctive 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. 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



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 reccommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.

References

Molecular Cloning: A Laboratory Manual, Vol 1.

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