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Team:Newcastle/E. coli Competence
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Shethharsh08 (Talk | contribs) (→Materials required) |
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==Procedures== | ==Procedures== | ||
| - | # Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/ | + | # Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain. |
# Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells). | # Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells). | ||
# Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes. | # Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes. | ||
Revision as of 14:48, 29 July 2010
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Contents |
Competent cells preparation
Materials required
- Conical flask
- 300ml of LB broth
- 1/20 volume of an overnight culture of the desired strain
- Ice and ice bucket
- eppendorf tubes
- TFB1
- TFB2
- 1.5 ml Microfuge tubes
- Ethanol dry ice
- -80°C freezer
Preperation of TFB1
Preperation of TFB2
Procedures
- Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.
- Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
- Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
- Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
- Repeat the the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
- Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice
- Store at -80°C
   
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