Team:Wageningen UR/Protocol/DialysisCCMV

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(→Procedure)
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== Reagents & Materials ==
== Reagents & Materials ==
 +
 +
Reagents:
 +
<ul>
 +
<li>Disassembly buffer</li>
 +
<li>Disassembly buffer + 8M urea</li>
 +
<li>Dialysis buffer</li>
 +
<li>Reassembly buffer</li>
 +
</ul>
 +
 +
Materials:
 +
<ul>
 +
<li>Pipettes + pipettepoints</li>
 +
<li>Greinertubes</li>
 +
<li>Dialysis tubing</li>
 +
<li>Vortex</li>
 +
<li>French Press</li>
 +
<li>Centrifuge for Greiner tubes</li>
 +
<li>Sorval or a similar centrifuge</li>
 +
<li>Coldroom</li>
 +
</ul>
== Procedure ==
== Procedure ==

Revision as of 10:48, 10 September 2012


Dialysis of the VLPs

Reagents & Materials

Reagents:

  • Disassembly buffer
  • Disassembly buffer + 8M urea
  • Dialysis buffer
  • Reassembly buffer

Materials:

  • Pipettes + pipettepoints
  • Greinertubes
  • Dialysis tubing
  • Vortex
  • French Press
  • Centrifuge for Greiner tubes
  • Sorval or a similar centrifuge
  • Coldroom

Procedure

The temperature of the VLP’s is very important. All the work should be done in the cold room (4°C) and the used tubes have to be on ice.

  1. Resuspend the cells in 5 mL of Disassembly buffer
  2. Disrupt the cells by french press, cell pressure: 1000
  3. Add Disassembly buffer to 50 mL total volume
  4. Centrifuge the lysate until the insoluble fraction is pelleted – cold, 4700 rpm, 18 min
  5. Remove the supernatant completely and dissolve the pellet in 10 mL of cold Disassembly buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend the while cooling on ice. Do not allow the buffer to heat up above 25°C
  6. Allow the pellet to dissolve for 2-5 minutes
  7. Dilute the 10 mL of 8 M urea/ Disassembly buffer with 15 mL Disassembly buffer. If crystals form, the batch should be discarded
  8. Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)
  9. Transfer the supernatant to a clean 50 mL Greiner tube
  10. Prepare a piece of dialysis tubing with a large diameter by soaking it in cooking demiwater until it opens up
  11. Put a tight knot on one side and fill the tubing with the supernatant
  12. Knot the tube on the other side leaving a small air bubble inside
  13. Dialyse the tube (about 25 mL volume) against 1x Dialysis buffer for 4 hours or overnight
  14. Dialysis is performed 6 times against 1x Dialysis buffer (4h per dialysis, or overnight)
  15. Dialysis is performed 2 times against Reassembly buffer (4h per dialysis, or overnight)


Continue further for CCMV formation

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