User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/06/04

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Cell and Protein Extraction

  1. The solutions were centrifuged for 15 minutes at 4500 rpm at 4 degrees Celsius
  2. The pellet was collected and placed in a 50mL falcon tube
  3. The falcon tube was centrifuged at 4500 rpm for an additional 15 minute to precipitate the cells
  4. The black supernatant was removed (but not discarded) and placed into a 15mL falcon tube
  5. A small amount of 5mM Tris buffer was used to collect remaining traces of cells from the centrifuge tubes
  6. The pellet was then sonified
    1. 30 seconds signification followed by 30 seconds on ice until the pellet was loose and liquid
  7. Once liquefied, the black supernatant was put back in the tubes and the solution was centrifuged at 20,000rpm four 3 hours at 4C
  8. The supernatant was then placed in dialysis tubing
  9. The tube and its content were placed in 50mM Tris buffer in the cold room (50F) overnight to remove unwanted salts