User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/15

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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 225rpm
  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 removed from the incubator
  7. The solutions were centrifuged for 15 minutes at 4500rpm and the supernatant (clear broth) was discarded
  8. The pellets were combined into 50mL falcon tube
  9. Protease inhibitors were prepared
    1. 50mg of AEBSF . HCL [Pefabloc SC] (from Enzo life Sciences) were dissolved in 35mL 50mM Tris buffer solution
  10. The solution was used to collect the remaining cells from the centrifuge tubes
  11. The tubes containing the cells and true Tris solution were centrifuged for 5 minutes at 2500rpm
  12. The tubes were placed in the freezer