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Team:Wageningen UR/Protocol/RoundupHepB
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| - | Materials: | + | == Materials: == |
<ul> | <ul> | ||
| - | <li>Pipettes + | + | <li>Pipettes + Pipette tips</li> |
<li>Eppendorf tubes</li> | <li>Eppendorf tubes</li> | ||
<li>Greiner tubes</li> | <li>Greiner tubes</li> | ||
| Line 14: | Line 14: | ||
<ol> | <ol> | ||
<li>Cut the knot of the dialysis tubing and empty the content into a clean 50 mL Greiner tube</li> | <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 | + | <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> |
<li>Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample</li> | <li>Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample</li> | ||
| - | <li>Balance the vails with | + | <li>Balance the vails with reassembly buffer</li> |
| - | <li>Centrifuge at 45000 | + | <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>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>A transparent pellet should be visible</li> | ||
| - | <li>Resuspend/ dissolve the pellet in 200 uL of Formation Buffer</li> | + | <li>Resuspend / dissolve the pellet in 200 uL of Formation Buffer</li> |
</ol> | </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
- Cut the knot of the dialysis tubing and empty the content into a clean 50 mL Greiner tube
- Prepare a Centrikon T-1055 ultra-centrifugation vessel and use a TFT 65.13 rotor, fill the vails with 4 ml of the sample
- Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample
- Balance the vails with reassembly buffer
- Centrifuge at 45000 RPM for 3 h, remember the orientation of the vessel so you know where to look for the pellet
- Take a sample from the supernatant (for Western blotting) and decant the rest (supernatant also contains single subunits)
- A transparent pellet should be visible
- Resuspend / dissolve the pellet in 200 uL of Formation Buffer
