Media preparation
1. General Protocols For Minimal Media Protein Expression
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
Add any antibiotics used to select the strains properly after the medium temperature is blew 37 oC.
3. General Protocols For High-Cell-Density Media
For 1 L media preparation
50 mM Na2HPO4.7H2O
25 mM KH2PO4 (pH 8.0-8.2)
10 mM NaCl
5 mM MgSO4
0.2 mM CaCl2
0.1% NH4Cl or 15NH4Cl
1.0% Glucose or 13C-Glucose
To prepare the media, glucose and metal solution are filtered to sterilize. The salt solution is autoclaved and the cooled solution is added by metal solution and glucose.