IGEM:British Columbia/Protocols/Competent Cells

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Contents

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
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