Large Scale Growth
LB (4 L, in Fernback flasks):
10 g of Tryptone (or peptone), 5 g of yeast extract, 10 g of NaCl
Autoclave on liquid setting (autoclave program #7)
Day 1:
- Make LB (place flasks in 37C room overnight for faster growing tomorrow)
- Start overnight culture: 30 mL LB in sterile flask + 30 uL of antibiotic(s), inoculate with glycerol stock
Shake at 37C overnight (12-16 hours)
Day 2:
- For each 1 L flask of LB, add:
1 mL of antibiotic(s)
6.5 mL of overnight culture - Shake cultures in 37C, note start time
- Grow cells until absorbance (OD) at 600 nm is between 0.6-0.8 (if 1 L flasks are pre-warmed, it will not take longer than 3 hours)
- Induce cells to overexpress protein by adding IPTG to a final concentration of 0.4 mM (0.4 mL of 1 M IPTG)
- Grow cells in condition best for production of your protein:
- At 37C for 4 hours
- At room temp for 6 hours
- At 16C overnight
Spin at 6000 rpm for 15 minutes
Pour off supernatant, and repeat spins until all cells have been pelleted