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