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Expression Protocol in M9 Minimal Media via T7 Promoter

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: To prepare 500mL M9 minimal media;
  1. pH solution to 7.3 and filter sterilize (0.2u filter).

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

  3. Grow 5mL overnight culture in same media to inoculate 500mL M9.

  4. Shake culture at 37oC until an OD600 of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).