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>
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<li>Ice-cold 5mM MgSO4</li>
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<li>Ice-cold 5mM MgSO<sub>4</sub></li>
</ul>
</ul>
<h2>Protocol</h2>
<h2>Protocol</h2>
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<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 4C for 10 minutes at 4,000 rpm</li>
+
<li>Pellet cells at 4°C for 10 minutes at 4,000 rpm</li>
-
<li>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</li>
+
<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

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

  1. Culture and induce protein-producing bacteria
  2. Spin down the cells for 10 min at 4,000 rpm
  3. 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
  4. Pellet cells at 4°C for 10 minutes at 4,000 rpm
  5. 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
  6. Spin at >13,000 rpm for 10 minutes. Carefully remove the supernatant and transfer to new tube
  7. Do SDS PAGE Gel analysis and store as required

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