Day 1

• Stab frozen stock of BL21/JM109 (DE3) cells containing desired plasmid for protein expression with sterile pipette tip
• Innoculate 40 ml of LB (Luria-Bertani) Media modified with appropriate antibiotic (i.e. 40 µl of 100 mg/ml Ampicillin)
• Grow overnight at 37 °C, shaking at 250 rpm

Day 2

• Pellet cells at 3000 x g for 10 min at 4 °C
• Pour off supernatant and resuspend in an equivalent amount of M9 media w/ antibiotic
• Add 1 ml resuspended cells to 1 L M9 media
• Grow 1 L cultures at 37 °C, shaking at 250 rpm, until mid-log phase
• Take 250 µl sample, centrifuge, discard supernatant, resuspend in 50 µl 2x SDS gel loading buffer
• Add:
  • lysine, phenylalanine and threonine to 100 mg/L
  • isoleucine, leucine and valine to 50 mg/L
  • L-selenomethionine to 60 mg/L
• Grow for 15 min at 37 °C, shaking at 250 rpm
• Lower temperature to 20 °C
• Add 0.5 ml Isopropyl-beta-D-thiogalactopyranoside (IPTG, 1 M stock)
• Grow at 20 °C overnight, shaking at 250 rpm

Reagents

M9 minimal media

To 780 ml sterile deionized water (cooled to 50 °C or less) add:

• 200 ml 5x M9 Salts
• 2 ml sterilized 1 M MgSO4
• 20 ml filter sterilized 20% glucose
• 0.1 ml sterilized 1 M CaCl2

5x M9 Salts

To 1 L deionized water add:

• 64 g Na2HPO4•7H20 (33.9 g N2HPO4 anhydrous) (dibasic)
• 15 g KH2PO4 (monobasic)
• 2.5 g NaCl
• 5 g NH4Cl

The MgSO4 and CaCl2 solutions should be prepared separately, sterilized by autoclaving, and added after diluting the 5x M9 salts to 1 L with sterile deionized water