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From 2012.igem.org

Protocol for preparing competent cells

• Preparation of CaCl₂ buffer for competent cells!
• Before you start make sure that the <chemform>CaCl2</chemform> buffer is ice-cold when needed and the centrifuge is cooled to 4°C
• General procedure:

1. Inoculate LB-liquid medium with 1 mL of overnight culture
2. Let cells grow to OD600 0.4-0.6 (Li et al. 2010)
3. Transfer them to 50 mL Falcons and centrifuge for 5 min at 1500 g and 4 °C
4. Resuspend pellet in 5-10 mL <chemform>CaCl2</chemform> buffer; here 7 mL (prechilled)
5. Centrifuge 5 min 1500 g, 4 °C
6. Resuspend pellet in 5-10 mL <chemform>CaCl2</chemform> buffer; here 7 mL (prechilled)
7. Incubate cells 30-60 min on ice; here 30 min; Eppis prechilled in 5-10 mL on dry ice (or in liquid nitrogen)
8. Storage at -80°C

Literature

• Li et al. 2010.