Ampicillin

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==Mode of Action==
==Mode of Action==
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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.
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Inhibits the formation of cross-links in the peptidoglycan layer (which prvides 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==
==Mechanism of Resistance==

Revision as of 15:33, 24 January 2006

Contents

Mode of Action

Inhibits the formation of cross-links in the peptidoglycan layer (which prvides 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.

Plate color code

Orange stripe

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.

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

References

Molecular Cloning: A Laboratory Manual, Vol 1.

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