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Team:Calgary/Notebook/Protocols/AmpicillinSurvivalAssay2
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<h1>Ampicillin Survival Assay 2</h1> | <h1>Ampicillin Survival Assay 2</h1> | ||
| - | <p class="noIndent">We used this protocol to verify if, after protein purification, beta-lactamase was functional when linked to one of our TALEs.</p> | + | <p class="noIndent">We used this protocol to verify if, after protein purification, β-lactamase was functional when linked to one of our TALEs.</p> |
<h2>Reagents and Materials</h2> | <h2>Reagents and Materials</h2> | ||
<ul> | <ul> | ||
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<li>Bacteria resistant to chloramphenicol but not ampicillin</li> | <li>Bacteria resistant to chloramphenicol but not ampicillin</li> | ||
<li>Bacteria resistant to both ampicillin and chloramphenicol</li> | <li>Bacteria resistant to both ampicillin and chloramphenicol</li> | ||
| - | <li>Purified beta-lactamase protein</li> | + | <li>Purified β-lactamase protein</li> |
</ul> | </ul> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
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<li>Add your purified proteins in multiple tubes with different amounts of proteins; for construct we added the following: 20µg, 15µg, 10µg, 5µg, 1µg, 0.1µg and 0.01µg</li> | <li>Add your purified proteins in multiple tubes with different amounts of proteins; for construct we added the following: 20µg, 15µg, 10µg, 5µg, 1µg, 0.1µg and 0.01µg</li> | ||
<li>Incubate the tubes for 1h at 37°C</li> | <li>Incubate the tubes for 1h at 37°C</li> | ||
| - | <li>For the positive control, inoculate a tube that | + | <li>For the positive control, inoculate a tube that does not have protein on it with a colony of your bacteria resistant to both antibiotics</li> |
<li>Add colonies of your bacteria resistant to chloramphenicol but not ampicillin to the other tubes</li> | <li>Add colonies of your bacteria resistant to chloramphenicol but not ampicillin to the other tubes</li> | ||
<li>Grow the cells overnight</li> | <li>Grow the cells overnight</li> | ||
</ol> | </ol> | ||
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Latest revision as of 01:43, 29 October 2013
Ampicillin Survival Assay 2
Ampicillin Survival Assay 2
We used this protocol to verify if, after protein purification, β-lactamase was functional when linked to one of our TALEs.
Reagents and Materials
- LB Broth
- Ampicillin (100mg/mL)
- Chloramphenicol (30mg/mL)
- Falcon tubes
- Bacteria resistant to chloramphenicol but not ampicillin
- Bacteria resistant to both ampicillin and chloramphenicol
- Purified β-lactamase protein
Protocol
- To each Falcon tube, add 5mL of LB Broth, 5µL of ampicillin and 3µL of chloramphenicol
- Add your purified proteins in multiple tubes with different amounts of proteins; for construct we added the following: 20µg, 15µg, 10µg, 5µg, 1µg, 0.1µg and 0.01µg
- Incubate the tubes for 1h at 37°C
- For the positive control, inoculate a tube that does not have protein on it with a colony of your bacteria resistant to both antibiotics
- Add colonies of your bacteria resistant to chloramphenicol but not ampicillin to the other tubes
- Grow the cells overnight
