Last Update: December 2006
1. Luria-Bertani (LB) media (1 L):
Mix 10 g of Bacto-tryptone, 5 of Yeast extract, and 10 g of NaCl (for taste). pH to 7.5 w/ NaOH. And dH2O to 1 L (Autoclave)
2. 1M CaCl2 (1 L):
Mix 111 g of CaCl2 (anhydrous) and 1 L of dH2O. Filter sterilize through a 0.22m filter
3. 0.1M CaCl2 (1 L):
Mix 100 mL of 1M CaCl2 with 900 mL of dH2O. Filter sterilize through a 0.22m filter
4. 50% Glycerol (500 mL):
Mix 50 mL of Glycerol with 50 mL of dH2O (Autoclave)
5. 0.1M CaCl2 + 15% glycerol:
Mix 100 mL of 1M CaCl2, 300 mL of 50% Glycerol, and 600 mL of dH2O
6. LB plates:
Mix 500 mL of LB media with 7 g of Agar (Autoclave). Cool to ~55-65oC prior to pouring. The addition of antibiotics should be made before pouring and at a temperature not higher than 55oC. Antibiotics can also be spread on previously made plates, but this is not very effective (unequal absorption, etc...)
1. Streak E.coli cells (DH5a, HB101, GM8) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)
2. Allow cells to grow at 37oC overnight
3. Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37oC

4. Take 2 ml LB media and save for blank. Transfer 5 mL overnight DH5a culture into 500 mL LB media in 3 L flask

5. Allow cell to grow at 37oC (250 rpm), until OD600= 0.4 (~2-3 hours)
6. Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 20 mins
7. Centrifuge cells in Sorval GSA rotor at 4oC for 10 mins at 3,000 g
Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room

8. Pour off media and resuspend cells in 30 mL of cold 0.1 M CaCl2. Transfer the suspended cells into 50 mL polypropylene falcon tubes, and incubate on ice for 30 mins

9. Centrifuge cells using Sorval RT6000B rotor at 4oC for 10 mins at 3,000 g (2500 rpm)
10. Pour supernatant and resuspend cells (by pipetting) in 8 mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 mL into (1.5 mL) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months
NOTE: through the process, cells should be treated with care. No vortexing or excess pipetting should be performed, specially when the cells have been resuspended in CaCl2 because lysis will result, decreasing the amount of competent cells). Also, depending on the density of the cells, higher or lower volumes CaCl2 can be used to increase the concentration of cells per tube.

A similar protocol has been described using MgCl2. See Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. p.1.74.

Sources of commercial competent cells include: Clontech, Novagen (their NovaBlue Singles are excellent), and Invitrogen (One Shot competent cells)