"

From 2012.igem.org

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)

Next: CCMV purification