For initial conditions we recommend using rich media at 30°C. Otherwise, overnight at 16°C is possible.
At 30°C or 16°C, inoculate 1% overnight culture and grow cells at 30°C for 3 hours until OD₆₀₀ ~ 0.8 and then induce expression of protein for at least 5 hours at 30°C or overnight at 16°C.
If using 37°C, inoculate 1% overnight culture and grow cells for 2 hours at 37°C until OD₆₀₀~0.8 and then induce expression of protein for at least 6 hours at 37°C.