Before starting:

• Prepare all required reagents (SOB medium, sterile 10% glycerol)
• Make sure you have access to a pre-cooled centrifuge (4°C) and liquid nitrogen
• Prepare sterile, pre-cooled centrifuge tubes (4°C) and 1.5 ml Eppendorf tubes

Protocol:

1. Inoculate 30 mL SOB with a single colony from a fresh plate and grow the culture overnight at 37°C
2. Inoculate 1 L SOB with 10 ml of the preculture (use a ratio of 1:100 for the inoculation) and grow it at 37°C, 200 rpm until the OD₆₀₀ ~ 0.4-0.5 (takes approximately 4 h); continue working on ice or at 4°C for all steps from here on!
3. Centrifuge the cells at 4000 rpm / 20 min / 4°C
4. Wash the cell pellet with 1 volume of sterile 10 % glycerol
5. Centrifuge the cells at 4000 rpm / 20 min / 4°C
6. Wash the cell pellet with 0.5 volumes of sterile 10 % glycerol
7. Centrifuge the cells at 4000 rpm / 20 min / 4°C
8. Wash the cell pellet with 0.1 volumes of sterile 10 % glycerol
9. Centrifuge the cells at 4000 rpm / 20 min / 4°C
10. Add 0.5 – 1 mL of sterile 10 % glycerol (per liter of liquid culture you started with); resuspend the cell pellet
11. Prepare 100 µl aliquots in 1.5 mL Eppendorf tubes and freeze them immediately in liquid nitrogen
12. Store the electrocompetent cells at -80°C

SOB medium (per liter):

• 20 g Bacto Tryptone
• 5 g Bacto Yeast Extract
• 8.6 mM NaCl
• 2.5 mM KCl

• pH 7.0
• auto-clave
• add sterile 10 mM MgCl₂ and 10 mM MgSO₄