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Team:Newcastle/E. coli Competence
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| - | = | + | =Preparing competent ''E. coli''= |
==Materials required== | ==Materials required== | ||
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# Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain. | # 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 | + | # Grow the cell at 37°C, 200 rpm in an orbital incubator. |
# 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. | ||
# Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice. | # Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice. | ||
| - | #Repeat | + | # Repeat 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 | + | # 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 | + | # Store at -80°C. |
Latest revision as of 13:50, 27 October 2010
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Contents |
Preparing competent E. coli
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
Preparation of 100 ml of TFB1
| Number | Chemicals required | Volume |
|---|---|---|
| 1 | KAc | 30 mM |
| 2 | CaCl2 | 10 mM |
| 3 | KCl | 100 mM |
| 4 | Glycerol | 15% (v/v) |
| 5 | Distill water | 900 ml |
| 6 | 500 mM MnCl2.4H2O | 100 ml |
Preparation of 100 ml of TFB2
| Number | Chemicals required | Volume |
|---|---|---|
| 1 | CaCl2 | 75 mM |
| 2 | KCl | 10 mM |
| 3 | Glycerol | 15% (v/v) |
| 4 | Distill water | 900 ml |
| 5 | Na-MOPS pH 7.0 | 100 mM |
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, 200 rpm in an orbital incubator.
- 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 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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