Biology Protocols

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Preparation of competent cells Calcium Chloride method

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1. Inoculate 10mL of LB media with a colony of bacteria to be transformed and incubate overnight at 37ºC.

2. Aliquot 200μl of the overnight culture to 20mL of LB media and incubate at 37ºC until the O.D.600nm is ~0.3.

3. Harvest the cells by centrifugation at 3,000 x g for 10 minutes and discard the supernatant.

4. Resuspend the cells in 10mL of cold 50mM CaCl2 and leave on ice for 1 hour.

5. Centrifuge for 10 minutes at 3,000 x g.

6. Resuspend the pellet with 1-2mL of cold 50mM CaCl2 and either transform DNA straight away or leave on ice and use within 24 hours.

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