Team:Calgary/Notebook/Protocols/GlassBeadsCellLysisProtocolforProteinSamples

From 2013.igem.org

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<h2>Protocol</h2>
<h2>Protocol</h2>
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<ol>
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<li>Culture and induce protein-producing bacteria</li>
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<li>Culture and induce protein-producing bacteria.</li>
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<li>Centrifuge cells at max for 10 minutes. Discard supernatant</li>
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<li>Centrifuge cells at max for 10 minutes. Discard supernatant.</li>
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<li>Resuspend cells in 2mL of lysis buffer</li>
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<li>Resuspend cells in 1:10 dilution of lysis buffer (Lysis buffer composed of: 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0).</li>
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<li>In a 1.5mL tube, add ~0.5mL of glass beads and 1mL of cells resuspended in lysis buffer</li>
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<li>In a 1.5mL tube, add ~0.5mL of glass beads and 1mL of cells resuspended in lysis buffer.</li>
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<li>Leave on ice for 5 min. Leave on BeadBeater or Vortex for 5 min. Repeat this cycle 6 times</li>
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<li>Leave on ice for 5 min. Leave on BeadBeater or Vortex for 5 min. Repeat this cycle 2 times.</li>
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<li>Centrifuge at max for 20 minutes</li>
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<li>6. Centrifuge at 14,000rpm for 20 minutes.</li>
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<li>Transfer supernatant (crude lysate) to a new tube and discard the glass beads</li>
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<li>Transfer supernatant (crude lysate) to a new tube and discard the glass beads.</li>
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Latest revision as of 01:22, 29 October 2013

Glass Beads Cell Lysis Protocol for Protein Samples

Reagents and Materials

  • 0.1mm diameter glass beads
  • Lysis buffer
  • Overnight induced cultures
  • 1.5mL tubes

Protocol

  1. Culture and induce protein-producing bacteria.
  2. Centrifuge cells at max for 10 minutes. Discard supernatant.
  3. Resuspend cells in 1:10 dilution of lysis buffer (Lysis buffer composed of: 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0).
  4. In a 1.5mL tube, add ~0.5mL of glass beads and 1mL of cells resuspended in lysis buffer.
  5. Leave on ice for 5 min. Leave on BeadBeater or Vortex for 5 min. Repeat this cycle 2 times.
  6. 6. Centrifuge at 14,000rpm for 20 minutes.
  7. Transfer supernatant (crude lysate) to a new tube and discard the glass beads.

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