Before starting:

• Prepare all required reagents and solutions

Protocol:

1. Transform the vector into an appropriate E. coli expression strain and plate out on minimal medium plates; incubate overnight at 37°C
2. Pick a colony and use this to inoculate 5 ml of Medium A; grow overnight at 37°C
3. Add the overnight culture to 1 liter of Medium A and grow the culture at the appropriate temperature until the OD₆₀₀ ~ 0.8 – 1.0
4. Induce expression of the target protein and continue culturing for 2-12 hours
5. Harvest the cells by centrifugation at 4°C
6. When you don’t continue immediately with the protein purification, freeze the cell pellet and store at -20°C until further usage

Comment: the growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector backbone, the construct design and the solubility of the protein of interest

Medium A (per liter):

• 100 ml M9 medium (10x)
• 10 ml trace elements solution (100x)
• 20 ml 20% (w/v) glucose
• 1 ml 1M MgSO₄
• 0.3 ml 1M CaCl₂
• 1 ml biotin (1 mg/ml)
• 1 ml thiamin (1 mg/ml)
• Appropriate antibiotics

10x M9 medium (per liter)

• 60 g Na₂HPO₄
• 30 g KH₂PO₄
• 5 g NaCl
• 5 g N¹⁵H₄Cl

100 x trace elements solution (per liter):

• 5 g EDTA
• 0.83 g FeCl₃.6H₂O
• 84 mg ZnCl₂
• 13 mg CuCl₂.2H₂O
• 10 mg CoCl₂.6H₂O
• 10 mg H₃BO₃
• 1.6 mg MnCl₂.6H₂O

Comment: First dissolve the 5 g EDTA in 800 ml water and adjust the pH to 7.5. Then add the other components and adjust the volume to 1 L. Sterilize the solution by filtration through a 0.22 µm filter.

Stock solutions

• 20% (w/v) glucose (sterilized)
• 1M MgSO₄ (sterilized)
• 1M CaCl₂ (sterilized)
• 1 mg/ml Biotin (filter sterilized)
• 1 mg/ml Thiamin (filter sterilized)