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Chemical Transformation of E. coli (heat-shock transformation)

  • Thaw frozen competent cells (50ul) on ice (~5 min)
  • Add DNA, then flick to mix
    • Usually 2-5ul for ligations, 1ul (less than 10ng) for completed constructs
    • Be careful not to warm cells
  • Incubate on ice for 30 minutes.
    • This is a minimum, can go longer
  • Heat shock for 45 seconds at 42°C.
  • Return cells to ice for 1-2 min
  • Recovery - add 900 ul of LB or SOB and incubate shaking for 1 hour at 37°C.
  • Plate some or all of the cells on LB Agar plates with appropriate antibiotic
  • If plating all of the cells
    • Briefly spin down the cells
      • 6000rpm for 2 minutes
    • Remove all but 50-100 ul of media.
    • Gently resuspend the cell pellet by pipetting
    • Plate on media containing the appropriate antibiotic.

- Remember, some expression strains have a resistance marker on a pre-existing plasmid; this antibiotic might need to be applied in order to maintain it.

- For transformations into expression strains (i.e. BL21 (DE3), 20-80 ul of cells (after the recovery incubation) can be directly added to a liquid O/N culture with antibiotics and then used to inoculate the larger scale expression culture the next day.