Step 1: Take an 100 mL aliquot of frozen cells from the -80^oC and innoculate about 500 ml to 1 L sterile LB broth. DO NOT ADD antibiotic, since these cells do not have an plasmid in them. Work as sterile as possible.

Step 2: Grow the cells on a shaker at 37^oC until they reach an OD @ 600nm of 0.3 to 0.4 (1 cm pathlength).

Step 3: Centrifuge in the Sorvall GSA rotor (250 ml centrifuge bottle) at 5,000 RPM for 10 minutes at 4^oC. Ice down 100 mM CaCl₂ and 100 mM MgCl₂ solutions at this point.

Step 4: Gently resuspend the bacteria pellet on ice in 1/4 volume of ice cold MgCl₂ , taking 3 to 5 minutes for this procedure. Centrifuge he cell suspension at 4,000 RPM in the Sorvall GSA rotor for 10 minutes.

Step 5: Resuspend the bacteria pellet on ice in 1/20 volume of ice cold CaCl₂ and then add an addional 9/20 volume of CaCl₂. Keep this suspension on ice for at least 20 minutes.

Step 6: Centrifuge the cell suspension at 4,000 RPM in the GSA rotor for 10 minutes and resuspend the cell pellet in 1/50 volume of ice cold, sterile  85 mM CaCl₂ in 15% glycerol w/v.  Dispense in 100 mL aliquots and freeze cells at -80^oC.