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From 2012.igem.org

Purification of the VLPs

Reagents & Materials

Reagents:

• Reassemblybuffer
• Virus buffer

Materials:

• Pipettes + Pipettepoints
• 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 ultracentrifugation 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 reasseblybuffer
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 Virus buffer