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 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==
==Mechanism of Resistance==
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Expression of [[&beta;-lactamase]] neutralizes ampicillin.  The resistance gene is named ''bla'' or ''amp<sup>R</sup>''.  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.
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Expression of [[&beta;-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.
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[[Image:Amp2.jpg|thumb|300px|left|Satellite colonies on an Ampicillin plate]]
[[Image:Amp2.jpg|thumb|300px|left|Satellite colonies on an Ampicillin plate]]
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==Stock Solution==
==Stock Solution==
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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.
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Typical concentrations of ampicillin are 50 μg/mL for low copy plasmids and 100 μg/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 55°C or below prior to adding antibiotic.
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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.
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==Stability==
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Culture plates with ampicillin can be stored at 2-8°C for up to two weeks. (In our experience, plates are usable for 2-3 months when stored at 4°C  and bagged to prevent evaporation. -[[User:Tk|tk]]) 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. [http://www.sigmaaldrich.com/sigma/product%20information%20sheet/a0166pis.pdf Sigma reference]
==Usage Notes==
==Usage Notes==
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A 1990 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2199796 paper] by [http://www.biology.bnl.gov/cellbio/studier.html Bill Studier] discusses how the secreted [[&beta;-lactamase]] can quickly consume all the ampicillin in a culture (even at 20&mu;g/ml Amp).  A stationary culture of ampicillin resistant cells can have such a concentration of [[&beta;-lactamase]] that even a 1/200 to 1/1000 dilution will still contain enough [[&beta;-lactamase]] to consume all the fresh ampicillin before all the non-resistant cells from the stationary phase culture have been killed.   
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A 1990 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2199796 paper] by [http://www.biology.bnl.gov/cellbio/studier.html Bill Studier] discusses how the secreted [[&beta;-lactamase]] can quickly consume all the ampicillin in a culture (even at 20&mu;g/ml Amp).  A stationary culture of ampicillin resistant cells can have such a concentration of [[&beta;-lactamase]] that even a 1/200 to 1/1000 dilution will still contain enough [[&beta;-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.
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*[[Barry Canton]] & [[Matt Gethers]] have seen this occur when using the BioBrick plasmid, pSB1A3 expressing a high level of mCherry.
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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.
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[[Carbenicillin]] is much more resistant and would be preferred except for cost.  Mixtures of ampicillin and carbenicillin are often used.
==References==
==References==
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Molecular Cloning: A Laboratory Manual, Vol 1.
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<biblio>
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#Anderson-Lancet-1965 pmid=14238093
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#Datta-BiochemJ-1966 pmid=5328167
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#Evans-JBacteriol-1968 pmid=4971890
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#MolecularCloning isbn=0-87969-577-3
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</biblio>
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[[Category:Material]] [[Category:Antibiotic]]

Current revision

Contents

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 μg/mL for low copy plasmids and 100 μg/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 55°C 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.

Stability

Culture plates with ampicillin can be stored at 2-8°C for up to two weeks. (In our experience, plates are usable for 2-3 months when stored at 4°C and bagged to prevent evaporation. -tk) 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.

References

Error fetching PMID 14238093:
Error fetching PMID 5328167:
Error fetching PMID 4971890:
  1. Error fetching PMID 14238093: [Anderson-Lancet-1965]
  2. Error fetching PMID 5328167: [Datta-BiochemJ-1966]
  3. Error fetching PMID 4971890: [Evans-JBacteriol-1968]
  4. isbn:0-87969-577-3. [MolecularCloning]
All Medline abstracts: PubMed HubMed
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