IGEM:British Columbia/Protocols/Competent Cells

Competent Cell Preparation

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