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(Based on Inoue et al. (1990), Gene, 96:23-28)

  1. Grow cells (e.g. DH5 α) on LB agar plate at 37° C overnight.
  2. Inoculate pre-culture in 250ml SOB in a 1L flask, 18-20°C with vigorous shaking to OD600 ~ 0.4-0.6 (normally it takes 24-36 hrs)
  3. Place the flask on ice for 10 min.
  4. Pellet the cells at 4000rpm for 10 min at 4° C.
  5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
  6. Spin at 4000rpm for 10 min at 4° C
  7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO, place on ice for 10 min
  8. Prepare 50-500µl aliquots, freeze immediately in liquid N2 and store at -80°C.
SOB Bacto Tryptone 20 g
  Bacto Yeast Extract 5 g
  8,6 mM NaCl 0,5 g
  2,5 mM KCl 2,5 mL (from 1 M stock)
  H2O ad 1000 mL
  pH = 7.0  
  autoclave and add sterile  
  10 mM MgCl2 5 mL (from 2 M stock)
  10 mM MgSO4 5 mL (from 2 M stock)
TB solution 10mM PIPES 116 mL
  250mM KCl  
  15mM CaCl2  
  Adjust pH to 6.7 with KOH  
  Add MnCl2 to 55mM, and adjust to final volume.
  Sterilize by filtration with 0.45µm filter and at 4° C