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Team:Wageningen UR/Protocol/DialysisPolero
From 2012.igem.org
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| - | <li>Resuspend the cells in 5 mL of | + | <li>Resuspend the cells in 5 mL of Formation buffer</li> |
<li>Disrupt the cells by french press, cell pressure: 1000</li> | <li>Disrupt the cells by french press, cell pressure: 1000</li> | ||
| - | <li>Add | + | <li>Add Formation buffer to 25 mL total volume</li> |
<li>Centrifuge the lysate until the insoluble fraction is pelleted – 4°C, 4700 rpm, 18 min</li> | <li>Centrifuge the lysate until the insoluble fraction is pelleted – 4°C, 4700 rpm, 18 min</li> | ||
| - | <li>Remove the supernatant completely and dissolve the pellet in 10 mL of cold | + | <li>Remove the supernatant completely and dissolve the pellet in 10 mL of cold Formation buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend it while cooling on ice. Do not allow the buffer to heat up above 25°C</li> |
<li>Allow the pellet to dissolve for 2-5 minutes</li> | <li>Allow the pellet to dissolve for 2-5 minutes</li> | ||
| - | <li>Dilute the 10 mL of 8 M urea/ | + | <li>Dilute the 10 mL of 8 M urea/ Formation buffer with 15 mL Formation buffer. If crystals form, the batch should be discarded</li> |
<li>Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)</li> | <li>Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)</li> | ||
<li>Transfer the supernatant to a clean 50 mL Greiner tube</li> | <li>Transfer the supernatant to a clean 50 mL Greiner tube</li> | ||
| Line 42: | Line 42: | ||
<li>Put a tight knot on one side and fill the tubing with the supernatant</li> | <li>Put a tight knot on one side and fill the tubing with the supernatant</li> | ||
<li>Knot the tube on the other side leaving a small air bubble inside</li> | <li>Knot the tube on the other side leaving a small air bubble inside</li> | ||
| - | <li>Dialyse the tube (about 25 mL volume) against 1x | + | <li>Dialyse the tube (about 25 mL volume) against 1x Formation buffer for 4 hours or overnight</li> |
<li>Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)</li> | <li>Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)</li> | ||
</ol> | </ol> | ||
Revision as of 08:22, 18 October 2012
Dialysis of the VLPs
This protocol is based on the CCMV VLP formation protocol. We made adjustments because Polero has a different pH at which the particle is stable. Besides, the number of dialysis steps is reduced and only 1 buffer is needed for the dialysis.
Reagents & Materials
Reagents:
- Formation Buffer
- Formation Buffer + 8M urea
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.
- Resuspend the cells in 5 mL of Formation buffer
- Disrupt the cells by french press, cell pressure: 1000
- Add Formation buffer to 25 mL total volume
- Centrifuge the lysate until the insoluble fraction is pelleted – 4°C, 4700 rpm, 18 min
- Remove the supernatant completely and dissolve the pellet in 10 mL of cold Formation buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend it while cooling on ice. Do not allow the buffer to heat up above 25°C
- Allow the pellet to dissolve for 2-5 minutes
- Dilute the 10 mL of 8 M urea/ Formation buffer with 15 mL Formation buffer. If crystals form, the batch should be discarded
- Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)
- Transfer the supernatant to a clean 50 mL Greiner tube
- Prepare a piece of dialysis tubing with a large diameter by soaking it in cooking demiwater until it opens up
- Put a tight knot on one side and fill the tubing with the supernatant
- Knot the tube on the other side leaving a small air bubble inside
- Dialyse the tube (about 25 mL volume) against 1x Formation buffer for 4 hours or overnight
- Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)
Continue further for Polero formation
