M9 Minimal Medium

1M9 Minimal Medium
Per liter: Na₂HPO₄­·7H₂O 12.8g

OR

Na₂HPO₄ (anhydrous) 6 g

KH₂PO₄ 3 g

NaCl 0.5 g

NH₄Cl 1 g
-add a carbon source (glucose, sodium gluconate, or glycerol) to 0.2% (v/v) final volume

-pH to 7.4 with NaOH

-Autoclave and then add sterile micronutrient components to a final concentration of:
Stock Concentration Micronutrient Final Concentration

1 M MgSO₄ 1 mM

1 M CaCl₂ 100 M

3 mM (NH₄)₆Mo₇O₂₄·4H₂O 3x10^-9 M

400 mM H₃BO₃ 4x10^-7 M

30 mM CoCl₂ ·6H₂O 3x10^-8 M

10 mM CuSO₄ ·5H₂O 1x10^-8 M

80 mM MnCl₂ ·4H₂O 8x10^-8 M

10 mM ZnSO₄ ·7H₂O 1x10^-8 M

5 mM FeSO₄ ·7H₂O§ 1x10^-6 M

• 
  I recommend filter sterilization for all micronutrients
  
• 
  §FeSO₄ must be made fresh and filter sterilized immediately before use. This solution is extremely prone to oxidation and precipitation, but may be frozen in aliquots.
  

Alternatively, make a 10X solution of the M9 salts, a 1000X mixed solution of the (NH₄)₆Mo₇O_24, H₃BO₃ , CoCl₂ , CuSO_4, MnCl₂, and ZnSO₄ and mix that with the appropriate FeSO₄, MgSO₄, CaCl₂, and carbon source (with any antibiotics, arabinose, etc., etc.)

For 250mL of M9 solid medium (*autoclaved separately)
Component Volume Final concentration

1.5% (w/v) agar in MQ water* 200 mL 1% (w/v)

10X M9 salts* 25 mL 1X

20% (w/v) carbon source 2.5 mL 0.2% (w/v)

1000X micronutrients 0.25 mL 1X

1M MgSO₄ 0.25 mL 1 mM

1M CaCl₂ 0.025 mL 100 μM

5 mM FeSO 0.05 mL 1 μM

Milli-Q water* 21.925 mL

250mL