Difference between revisions of "Ampicillin"

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==Mode of Action==
 
==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.
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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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==Stock Solution==
 
==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.
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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.
  
==Plate color code==
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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.
Orange stripe
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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==
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 [[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.   
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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 [[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.
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*[[Barry Canton]] & [[Matt Gethers]] have seen this occur when using the BioBrick plasmid, pSB1A3 expressing a high level of mCherry.
  
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.
 
[[Carbenicillin]] is much more resistant and would be preferred except for cost.  Mixtures of ampicillin and carbenicillin are often used.
  
 
==References==
 
==References==
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]]

Latest revision as of 20:46, 9 February 2008

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


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

  1. ANDERSON ES and DATTA N. RESISTANCE TO PENICILLINS AND ITS TRANSFER IN ENTEROBACTERIACEAE. Lancet. 1965 Feb 20;1(7382):407-9. PubMed ID:14238093 | HubMed [Anderson-Lancet-1965]
  2. Datta N and Richmond MH. The purification and properties of a penicillinase whose synthesis is mediated by an R-factor in Escherichia coli. Biochem J. 1966 Jan;98(1):204-9. PubMed ID:5328167 | HubMed [Datta-BiochemJ-1966]
  3. Evans J, Galindo E, Olarte J, and Falkow S. Beta-lactamase of R factors. J Bacteriol. 1968 Oct;96(4):1441-2. PubMed ID:4971890 | HubMed [Evans-JBacteriol-1968]
  4. ISBN:0-87969-577-3 [MolecularCloning]
All Medline abstracts: PubMed | HubMed