Team:Wageningen UR/Protocol/RoundupHepB

From 2012.igem.org

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== Materials: ==
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<ul>
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<li>Pipettes + Pipette tips</li>
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<li>Eppendorf tubes</li>
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<li>Greiner tubes</li>
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<li>Centrikon or a similar ultracentrifuge + all additional equipment</li>
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</ul>
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== Procedure ==
 +
 +
<ol>
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<li>Cut the knot of the dialysis tubing and empty the content into a clean 50 mL Greiner tube</li>
 +
<li>Prepare a Centrikon T-1055 ultra-centrifugation vessel and use a TFT 65.13 rotor, fill the vails with 4 ml of the sample</li>
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<li>Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample</li>
 +
<li>Balance the vails with reassembly buffer</li>
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<li>Centrifuge at 45000 RPM for 3 h, remember the orientation of the vessel so you know where to look for the pellet</li>
 +
<li>Take a sample from the supernatant (for Western blotting) and decant the rest (supernatant also contains single subunits)</li>
 +
<li>A transparent pellet should be visible</li>
 +
<li>Resuspend / dissolve the pellet in 200 uL of Formation Buffer</li>
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</ol>

Latest revision as of 01:04, 27 September 2012


Materials:

  • Pipettes + Pipette tips
  • Eppendorf tubes
  • Greiner tubes
  • Centrikon or a similar ultracentrifuge + all additional equipment

Procedure

  1. Cut the knot of the dialysis tubing and empty the content into a clean 50 mL Greiner tube
  2. Prepare a Centrikon T-1055 ultra-centrifugation vessel and use a TFT 65.13 rotor, fill the vails with 4 ml of the sample
  3. Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample
  4. Balance the vails with reassembly buffer
  5. Centrifuge at 45000 RPM for 3 h, remember the orientation of the vessel so you know where to look for the pellet
  6. Take a sample from the supernatant (for Western blotting) and decant the rest (supernatant also contains single subunits)
  7. A transparent pellet should be visible
  8. Resuspend / dissolve the pellet in 200 uL of Formation Buffer
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