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:

• 64g Na₂HPO₄-7H₂O
  
• 15g KH₂PO₄
  
• 2.5g NaCl
  
• H₂O to final 1L volume and autoclave

To prepare 500mL M9 minimal media;

• 100mL of 5xM9 salts
  
• 1 mL 1M MgSO₄
  
• 50 uL 1M CaCl₂
  
• 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
  
• 2.5 mL filter sterilized NH₄Cl (0.2 g/mL) or 0.5g dry
  
• 10 mL 20% d-glucose or 2g dry
  
• glass distilled & autoclaved H₂O to final volume of 500mL

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