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Team:Calgary/Notebook/Protocols/OsmoticShock
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<li>Induced cultures of protein-producing bacteria</li> | <li>Induced cultures of protein-producing bacteria</li> | ||
<li>20% w/v sucrose, 0.03 M Tris-HCl pH 8.0</li> | <li>20% w/v sucrose, 0.03 M Tris-HCl pH 8.0</li> | ||
| - | <li>Ice-cold 5mM | + | <li>Ice-cold 5mM MgSO<sub>4</sub></li> |
</ul> | </ul> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| Line 18: | Line 18: | ||
<li>Spin down the cells for 10 min at 4,000 rpm</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 | + | <li>Pellet cells at 4°C for 10 minutes at 4,000 rpm</li> |
| - | <li>Re-suspend pellet in 200µL of ice-cold 5mM | + | <li>Re-suspend pellet in 200µL of ice-cold 5mM MgSO<sub>4</sub> and transfer into an eppendorf tube and let sit on ice bath for 30 minutes</li> |
<li>Spin at >13,000 rpm for 10 minutes. Carefully remove the supernatant and transfer to new tube</li> | <li>Spin at >13,000 rpm for 10 minutes. Carefully remove the supernatant and transfer to new tube</li> | ||
<li>Do <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/SDSPAGEGel" target="_blank">SDS PAGE Gel</a> analysis and store as required</li> | <li>Do <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/SDSPAGEGel" target="_blank">SDS PAGE Gel</a> analysis and store as required</li> | ||
Latest revision as of 00:27, 28 September 2013
Osmotic Shock
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 4°C 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
