Media preparation

1. General Protocols For Minimal Media Protein Expression

Expression Protocol in M9 Minimal Media via T7 Promoter: The following protocol has been used successfully to ¹⁵N or ¹³C/¹⁵N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:

64 g Na₂HPO₄-7H₂O 15 g KH₂PO₄ 2.5 g NaCl

H₂O to final 1L volume and autoclave To prepare 500 mL M9 minimal media:

100mL of 5xM9 salts 1 mL 1 M MgSO₄ 50 uL 1 M CaCl₂ 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco) 2.5 mL filter sterilized NH₄Cl (0.2 g/mL) or 0.5 g dry 10 mL 20% d-glucose or 2 g dry

Glass distilled & autoclaved H₂O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).

Grow 5mL overnight culture in same media to inoculate 500mL M9. Shake culture at 37 ^oC until an OD₆₀₀ of 0.7 ± 0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).

2. General Protocols For LB Media

For 1 L media preparation, 10 g Bacto-tryptone 5 g yeast extract 10 g NaCl

Sterilized by autoclave