User:Matt Hartings/Notebook/Photosynthesis/2012/06/29

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Objective

WT Hemoglobin expression

  1. Centrifuge starter cultures from yesterday (20 minutes, 4500rpm, 4C)
  2. Add antibiotic to large culture flasks (1mL of 100mg/mL ampicilin)
  3. Resuspend cell pellets from starter culture and add to large culture flasks
  4. Place large culture flasks in shaker/incubator 37C, 165 rpm
  5. Grow bacteria to an OD of 0.6 at 600nm. (I overshot again and they grew to 1.3)
  6. Induce expression (add 1mL of 0.1M IPTG to each flask)
  7. Add hemin (1 mL of 40mg/mL hemin in DMSO was added to each flask)
    1. Note: The DMSO was used this time on the suggestion of Jay Storz and his postdoc - email correspondence.
  8. Shake/incubate at 30C, 165rpm for 4 hours
  9. Harvest cells, centrifuge for 30 minutes at 4500rpm and 4C
  10. Resuspend cell pellet in 50mL of 25mM Tris, 1mM EDTA, 0.5mM DTT pH 8.3
  11. Place in -80C Freezer