User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/05/28

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Protein Expression

  1. Cells were placed in each of the 25mL Erlenmeyer flasks and left to grow overnight in a shaking incubator at 37C and 200rpm
  2. Each of these solutions was transferred to a 1L broth solution with 1mL of ampicilin (100mg/mL)
  3. The flasks were left to incubate for 3 hours at 37C on the shaking incubator at 130rpm
  4. After 4 hours, 1ml of Fe-protoporphyrin IX was added to each flask and the temperature was reduced to 30C and left to incubate for another 4 hours
    1. The Fe-protoporphyrin IX (Hemin) was made by dissolving 40mg of Fe-protoporphyrin IX in 1mL of DMSO
  5. 1mL of IPTG was also added to each flask
    1. 4mM of IPTG in H20 prepared immediately before adding to flasks
  6. After four hours, the flasks were placed in a cold room over night

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 tube
  3. A small amount of 5mM Tris buffer was used to collect remaining traces of cells from the centrifuge tubes
  4. The pellet was then sonified
    1. 30 seconds signification followed by 30 seconds on ice until the pellet was loose and liquid
  5. The solution was then placed in the freezer overnight