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Team:Calgary/Sandbox/Notebook/Protocols/OsmoticShock
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<h2>Protocol</h2> | <h2>Protocol</h2> | ||
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| - | <li>Spin down the cells for 10 min at 4,000 rpm<li> | + | <li>Culture and induce protein-producing bacteria</li> |
| + | <li>Spin down the cells for 10 min at 4,000 rpm</li> | ||
<li>Re-suspend pellet in 10 ml of 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0. Incubate for 10 minutes at room temperature</li> | <li>Re-suspend pellet in 10 ml of 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0. Incubate for 10 minutes at room temperature</li> | ||
<li>Pellet cells at 4C for 10 minutes at 4,000 rpm</li> | <li>Pellet cells at 4C for 10 minutes at 4,000 rpm</li> | ||
Revision as of 23:31, 22 September 2013
Osmotic Shock
Reagents and Materials
- Induced cultures of protein-producing bacteria
- 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0
- Ice-cold 5mM MgSO4
Protocol
- Culture and induce protein-producing bacteria
- Spin down the cells for 10 min at 4,000 rpm
- Re-suspend pellet in 10 ml of 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0. Incubate for 10 minutes at room temperature
- Pellet cells at 4C for 10 minutes at 4,000 rpm
- Re-suspend pellet in 200µL of ice-cold 5mM MgSO4 and transfer into an eppendorf tube and let sit on ice bath for 30 minutes
- Spin at >13,000 rpm for 10 minutes. Carefully remove the supernatant and transfer to new tube
- Do SDS PAGE Gel analysis and store as required
