Preparation of competent E. coli cells using CaCl2

PREPARATION OF COMPETENT E. COLI CELLS USING CACL2

Last Update: December 2006

PREPARE SOLUTIONS

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 ~55-65^oC prior to pouring. The addition of antibiotics should be made before pouring and at a temperature not higher than 55^oC. Antibiotics can also be spread on previously made plates, but this is not very effective (unequal absorption, etc...)

PROCEDURE

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 37^oC overnight

3. Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37^oC

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 37^oC (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 4^oC 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 4^oC 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 -80^oC 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 CaCl₂because 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)