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M9 
 Description: M9 Salts (49.4mM sodium phosphate, dibasic, 24.0mM potassium phosphate, monobasic, 9mM sodium chloride, 19mM ammonium chloride) 1.5% Agar, 2% Glucose, 3.3μM Thiamine, 0.5mM Magnesium, Sulfate.
Shipping Conditions: Ambient
Storage Conditions: Room Temperature

M9 is a mimimal, defined growth medium used for cultivating recombinant strains of Escherichia coli. The M9 minimal media will also support the growth of “wild-type” strains of E. coli. M9 Medium is useful for maintaining positive selective pressure on recombinant strains of E. coli with plasmids coding for the ability to produce essential substances such as amino acids or vitamins. M9 is also used to maintain stocks of F’-containing bacteria for use with M13 bacteriophage.

The M9 medium has the advantage of being inexpensive, low auto-fluorescence (when excited at 488nm) and low absorbance. M9 can be supplemented with specific amino acids or other metabolites to allow for selection of specific auxotrophs (for example thiamine or casamino acids).

Sodium phosphate and potassium phosphate are present in M9 as buffering agents. Ammonium chloride provides a source of nitrogen for cells. Sodium chloride provides essential ions and osmotic support. Glucose or Glycerol may be added as a carbon source, to supply carbohydrates. Supplementing M9 with magnesium and calcium will increase the growth of recombinant E. coli.

Formula for 1X M9 (per liter):
0.3% (22mM) Potassium Phosphate (KH₂PO₄) (3.0g/L)
0.6% (22mM) Sodium Phosphate (Na₂HPO₄) (6.0g/L)
0.5% (85mM) Sodium chloride (NaCl) (5.0g/L)
0.1% Ammonium Chloride (NH₄Cl)(1.0g/L)
2mM Magnesium Sulfate (MgSO₄)
0.1mM Calcium Chloride (CaCl₂) (15.0mg/L) (optional)
0.4% Glucose (or Glycerol) (4.0g/L) (optional)

Alternately, 1X M9 Media can be made directly from 5X M9 salts stock solution:
5X M9 salts:
1.5% Potassium Phosphate (KH₂PO₄) (15.0g/L)
6.4% Sodium Phosphate (Na₂HPO₄) (64.0g/L)
0.25% Sodium chloride (NaCl) (2.5g/L)
0.5% Ammonium Chloride (NH₄Cl)(5.0g/L)
dissolved to final volume of 1 liter of water and autoclave to sterilize.

1X M9 (from 5X M9 salts):
200ml 5X M9 salts
2mM Magnesium sulfate (MgSO₄) (2ml of 1M MgSO₄)
0.1mM Calcium Chloride (CaCl₂) (0.1ml of 1M CaCl₂)
0.4% Glucose or Glycerol (20ml of 20%, for Carbon source)
Supplement with appropriate amino acids and vitamins, as necessary

Note: For E. coli strains with a deletion in proline biosynthetic operon [Δ(lac-proAB)] in the bacterial chromosome and the complementing proAB genes on the F’ plasmid it is necessary to supplement M9 minimal media:
0.4% (w/v) Glucose (Dextrose)
5mM MgS04
0.01% Thiamine

References:
1. Sambrook and Russell, Molecular Cloning, A Laboratory Manual, Volume 3 (2001).
2. Green and Sambrook, Molecular Cloning, A Laboratory Manual, Volume 4 (2012).