• start a 5 mL overnight culture of E. coli cells
• inoculate 100 mL 2 x TY to OD₆₀₀ = 0.1
• grow at 37 °C to OD₆₀₀ = 0.5 (in 1 liter flask)
• add 400 mL 2 x TY
• grow at 37 °C to OD₆₀₀ = 0.5
• put flask on ice in cold room
• cool down and shake from time to time
• pellet cells at 5000 rpm for 10 min, 4 °C
• resuspend in 100 mL Tfb-I
• incubate 10 min on ice
• spin down at 4000 rpm for 5min, 4 °C
• resuspend carefully in 20 mL Tfb-II
• aliquot into precooled 1.5 mL tubes (100-300µL/tube)
• freeze in liquid nitrogen, transfer to -80 °C
  
• Use 100 µL/transformation

Buffers:

 Tfb-I:
30 mM KOAc; 50 mM MnCl₂; 100 mM KCl; 10 mM CaCl₂; 15 % (w/v) Glycerin.

 Tfb-II:
10 mM Na-MOPS pH 7.0; 75 mM CaCl₂; 10 mM KCl; 15 % (w/v) Glycerin.