Per liter:

Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl (This solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H₂O). Adjust the pH to 7.0 with 5 N NaOH (~ 0.2 ml). Adjust the volume of the solution to 1 liter with deionized H₂O. Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle. Just before use, add 5 ml of a sterile solution of 2M MgCl₂

2. Sterile 2M MgCl₂ - Dissolve 19 g of MgCl₂ in 90 ml of deionized H₂O. Adjust the volume of the solution to 100 ml with deionized H₂O and sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle).

3. Sterile 10% Glycerol - 10% glycerol in distilled H₂O. Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle

1. Use a fresh colony of cells to inoculate 10 ml of SOB (without magnesium) in a 500 ml flask. Grow cells with vigorous aeration overnight at 37°C.

2. Dilute 0.5 ml of cells in 500 ml of SOB (without magnesium) in a 2.8 L flask. Grow for 2-3 h with vigorous aeration at 37°C until OD₅₅₀ = 0.8.
   Note: Higher efficiency cell preps can be obtained by doubling the initial culture volume (i.e. two 500 ml cultures) and pooling them during the first wash (Step 4).

3. Harvest cells by centrifuging at 5,000 rpm (2,600 x g) in a GSA or GS3 rotor for 10 min.

4. Wash the cell pellet by resuspending in 500 ml of sterile ice-cold 10% glycerol. Centrifuge the cell suspension at 5,000 rpm (2,600 x g) for 15 min and carefully pour off the supernatant as soon as the rotor stops.
   Note: Cells washed in 10% glycerol do not pellet well. If the supernatant is turbid, increase the centrifugation time.

5. Wash the cell pellet a second time by resuspending in 500 ml of sterile ice-cold 10% glycerol. Centrifuge the cell suspension at 5,000 rpm (2,600 x g) for 15 min, and pour off and carefully remove as much of the supernatant as possible.

6. Resuspend the cell pellet in ice-cold 10% glycerol to a final volume of 2 ml. Usually, no additional 10% glycerol needs to be added to the cell pellet; it can be resuspended in the 10% glycerol that remains in the centrifuge bottle. Cells can be used immediately or can be frozen in 20 µl aliquots in microcentrifuge tubes using a dry ice-ethanol bath (it is useful to prepare 40 µl, 80 µl, or 100 µl aliquots also). Store frozen cells at -70°C.
   Note: A greater volume of lower-efficiency cell preps can be obtained by resuspending the cells in ice-cold 10% glycerol to 10 ml instead of 2 ml. These cells, which typically will yield five- to tenfold fewer transformants/µg, may be frozen in 0.5 ml aliquots.