The following is the recipe to prepare ~ 1.0L of minimal salts medium for growth of prototrophic strains of E. coli.
The following are added to 900 mL of autoclaved milliQ water contained in a 2.7L Fernbach flask.
NOTE: Follow the order of addition given below. Also, be sure to mix the solution thoroughly after each addition.
1. 100 mL of 10X M9 salts (60g/L Na2HPO4, 30 g/L KH2PO4, 5 g/L NaCl, pH 7.4, autoclave for sterilization).
2. 10 mL of 100 g/L NH4Cl, pH 7.4 (use 0.22 mM filter for sterilization (since the ammonia is somewhat volatile, autoclaving is not recommended)).
3. 2.0 mL of 1.0 M MgSO4 (autoclave for sterilization).
4. 12.5 mL 20 % (w/v) D-glucose (autoclave for sterilization).
5. 0.2 mL 0.50 M CaCl2 (autoclave for sterilization).
6. Appropriate antibiotics, e. g. AMP at 100 mg/mL (use 0.22 mM filter for sterilization)
7. 1 mg biotin (added directly)
8. 0.5 mL 2 mg/mL thiamine hydrochloride (use 0.22 mM filter for sterilization)
9. 1.0 mL 15 mg/mL FeCl2 in 1.0 M HCl (use 0.22 mM filter for sterilization)
10. 1.0 mL 15 mg/mL ZnCl2 in slightly acidic H2O (add 2 – 3 drops 6.0 M HCl for 10 mL solution, use 0.22 mM filter for sterilization)
11. 2.0 mL 10% (w/v) yeast extract (autoclave for sterilization).
At this point, check the pH of the medium by swabbing a glass pH electrode with 100% Ethanol, and then by adjusting the pH to 7.40 using stocks of 6.0 M HCl and 6.0 M NaOH, as needed.
In order to facilitate the preparation of 15N-lableled proteins, the UT Health San Antonio general stores now carries 15NH4Cl as a item (July, 2003). It is sold in 1 gram bottles and the order ID is 175013787. The cost is roughly $35 per gram. 13C D-glucose is not available from stores, but can be pruchased through one of the stable isotope suppliers. The cost is roughly $90 per gram and the item numbers are: Cambridge CLM-1396-0 or Sigma-Aldrich 389374. For 13C D-glucose or if you need quantities of 15NH4Cl larger than about 5 grams, contact a one of the stable isotope suppliers