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
• After protein expression period is over, turn on centrifuge and spin at 1000 rpm for 10 minutes to let it cool down
• Harvest cells: pour into 300 mL centrifuge bottles and make sure each pair is balanced.
  Spin at 6000 rpm for 15 minutes
  Pour off supernatant, and repeat spins until all cells have been pelleted
• Scrape the pellets into 50 mL tubes (three centrifuge bottles for one 50 mL tube) and weigh cell paste, label tubes
• Freeze cell paste at -80C until ready for protein purification