BMBMethod:ChemicalTransformation

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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.