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Preparation of Chemically Competent Cells

Adapted from Inoue et al. 1990
Capable of ~3x109 cfu/ug

  1. Inoculate E. coli into 150mL SOB or LB media
  2. Grow between 18°C and 25°C until an OD600 of 0.5-0.6 is reached (overnight more convenient)
    1. At 18°C, it can certainly take 48+ hrs.
  3. Cool flask of cells on ice for 10 minutes
  4. Pellet cells by centrifugation at 4°C
  5. Gently resuspend pellet in 40mL of ice-cold TB
  6. Incubate cells on ice for 10 min
  7. Pellet cells by centrifugation at 4°C
  8. Resuspend cells in 5.58 mL of TB
  9. Add DMSO to final concentration of 7% (420 ul; 6mL final vol)
  10. Dispense into sterile microfuge tubes in 50-200ul aliquots
  11. Immediately snap freeze in liquid nitrogen
  12. Store in -80°C freezer until time to use

Transformation Buffer (TB) Recipe (50mL)

Reagent Amount Final Concentration
PIPES xxxg 10mM
CaCl2 xxxg 15mM
KCl xxxg 250mM
pH 6.7 (w/KOH) pH BEFORE adding MnCl2
MnCl2 xxxg 55mM


Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23–8. Retrieved from