IGEM:British Columbia/Protocols/Competent Cells

Materials

 * DH5 cells
 * LB broth (55mL)
 * 0.1M CaCl2 (filter sterilized and chilled)
 * 100% glycerol

Equipment

 * Spectrophotometer
 * 100-150mL flasks,
 * Chilled centrifuge bottles
 * Microfuge tubes
 * Ice water bath
 * Centrifuge

Method

 * Inoculate 5mL overnight culture and grow at 30°C
 * Dilute 0.5mL of overnight culture into 50mL LB and incubate at 30°C, shaking vigorously
 * Harvest cells at Abs600nm = 0.401
 * Centrifuge at 1600g for 7 minutes (4°C)
 * Wash gently in ~20mL COLD filter sterilized 0.1M CaCl2.
 * Spin down gently: 1100g, 5 minutes, (4°C)
 * Resuspend in 12.5mL cold 0.1M CaCl2
 * Keep on ice 40 minutes
 * Spin down: 1100g, 5 minutes, (4°C)
 * Resuspend in 2-2.5mL cold 0.1M CaCl2
 * Store over night at 4°
 * Add 100% glycerol to make a 14% glycerol solution
 * Make 100μL aliquots, store at -80°C