AU Biomaterials Design Lab:Protocols/Protein Expression: Difference between revisions
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(New page: ==Description== During protein expression, bacteria will be ["http://en.wikipedia.org/wiki/Bacterial_growth" grown] through log phase to near stationary phase. This will produce a maximum ...) |
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==Description== | ==Description== | ||
During protein expression, bacteria will be [ | During protein expression, bacteria will be [http://en.wikipedia.org/wiki/Bacterial_growth grown] through log phase to near stationary phase. This will produce a maximum number of bacteria which will synthesize our protein of interest while not depleting the nutrients in the broth. When the cultures reach this phase protein expression is induced and allowed to proceed for 3-4 hours. | ||
==Protocol== | ==Protocol== | ||
Latest revision as of 17:41, 11 August 2011
Description
During protein expression, bacteria will be grown through log phase to near stationary phase. This will produce a maximum number of bacteria which will synthesize our protein of interest while not depleting the nutrients in the broth. When the cultures reach this phase protein expression is induced and allowed to proceed for 3-4 hours.
Protocol
- The first steps need to happen first thing in the morning.
- Centrifuge the starter cultures at 4500rpm for 15 minutes
- Re-suspend the cell pellets in 4mL of fresh LB.
- Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.
- Incubate the expression cultures at 37C and 160rpm until the absorbance of the media at 600nm = 0.6.
- Add 1mL of 0.4M IPTG to each flask in order to induce protein expression
- Continue shaking for 3-4 hours
- Harvest the cells by centrifuging at 4500rpm for 15 minutes
- Collect the cells and place in the -80 freezer
