User:Matt Hartings/Notebook/Photosynthesis/2012/06/29: Difference between revisions
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==Objective== | ==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 | |||
Revision as of 12:15, 30 June 2012
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ObjectiveWT Hemoglobin expression
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