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Making Chemically Competent E. coli Cells

This procedure was done using Top10 cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.

  1. Innoculate 5-10 mL LB at 37°C while shaking
  2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37° 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. Resuspend 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 resuspend 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