Before starting:

• Prepare all required reagents and solutions
• 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. Streak out the desired bacterial cell line on an LB-agar plate and incubate overnight at 37°C
2. Inoculate 250 ml SOB and grow at 18-20°C, 200 rpm until the OD₆₀₀ ~ 0.4-0.6 (takes approximately 24-36 h); continue working on ice or at 4°C for all steps from here on!
3. Place the flask on ice for 10 min
4. Centrifuge the cells at 4000 rpm / 10 min / 4°C
5. Gently resuspend the cells in 80 ml ice-cold TB and keep on ice for 10 min.
6. Centrifuge the cells at 4000 rpm / 10 min / 4°C
7. Gently resuspend the cells in 20 ml ice-cold TB and 1.4 ml DMSO and keep on ice for 10 min.
8. Prepare 50 µl aliquots in 1.5 mL Eppendorf tubes and freeze them immediately in liquid nitrogen
9. Store the chemically competent 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

TB solution

• 10 mM PIPES
• 250 mM KCl
• 15 mM CaCl₂

• Adjust to pH 6.7 with KOH
• Add 55 mM MnCl₂
• Sterilize by filtration (0.45 µm filter) and store at 4°C