From 2010.igem.org
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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
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| 1
| KAc
| 30 mM
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| 2
| CaCl2
| 10 mM
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| 3
| KCl
| 100 mM
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| 4
| Glycerol
| 15% (v/v)
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| 5
| Distill water
| 900 ml
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| 6
| 500 mM MnCl2.4H2O
| 100 ml
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Preparation of 100 ml of TFB2
| Number
| Chemicals required
| Volume
|
| 1
| CaCl2
| 75 mM
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| 2
| KCl
| 10 mM
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| 3
| Glycerol
| 15% (v/v)
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| 4
| Distill water
| 900 ml
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| 5
| Na-MOPS pH 7.0
| 100 mM
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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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