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Team:TU Darmstadt/Notebook/Methods/Protein expression
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Revision as of 16:34, 17 October 2014
Protein expression
Equipment:
- Incubation shaker
- Photometer
Chemicals & consumables:
- E. coli BL21 DE3
- dYT medium or LB medium
- IPTG
- 100 ml and 3 l flasks
- Ice
Procedure:
Inoculation of 50 mL LB medium in a 100 mL flask with E. coli BL21 DE3 containing the plasmid with the sequence for the respective part to be expressed.
Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4
Transferation of the starter culture into 1 L LB medium in a 3 L flask resulting in an OD600= 0.2
Incubation to an OD600= 0.6 at 180 rpm and 30°C.
Incubation for 15 minutes on ice.
Induction of the protein expression with 20 mL of IPTG (stock concentration 1M).
Incubation of the cell suspension overnight at 180 rpm at 30°C.
