Team:Calgary/Notebook/Protocols/MakingChemicallyCompetentEcoliCells

From 2013.igem.org

Making Chemically Competent E. coli Cells

Reagents and Materials

  • Top10 E. coli cells
  • LB Broth
  • 50mM CaCl2 solution (store at 4°C)

Protocol

  1. Inoculate 5-10 mL LB at 37°C while shaking
  2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37°C; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
  3. Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
  4. Re-suspend pellet in 12.5 mL of cold CaCl2 (50 mM) and leave on ice for 10 minutes
  5. Centrifuge at 10 000 rpm at 4°C for 2 minutes and re-suspend in 2 mL of cold CaCl2 (50 mM, 15% glycerol solution)
  6. Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C