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Contents 1 Preparing competent E. coli 1.1 Materials required 1.1.1 Preparation of 100 ml of TFB1 1.1.2 Preparation of 100 ml of TFB2 1.2 Procedures

Preparing competent E. coli

Materials required

1. Conical flask
2. 300ml of LB broth
3. 1/20 volume of an overnight culture of the desired strain
4. Ice and ice bucket
5. eppendorf tubes
6. TFB1
7. TFB2
8. 1.5 ml Microfuge tubes
9. Ethanol dry ice
10. -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

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.
2. Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
3. Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
4. Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
5. Repeat the the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
6. Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice
7. Store at -80°C

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