TITLE: Standard M9 minimal medium
AUTHORS: Andreas Sagen

[DESCRIPTION]
M9 minimal media is a highly-referenced microbial growth medium used for the cultivation of E. coli. This buffered minimal microbial medium contains only salts and nitrogen, so it is traditionally supplemented with glucose, amino acids and vitamins as needed.
M9 Minimal Medium is suitable for non-selective cultivation of Escherichia coli strains for cloning, DNA plasmid production and production of recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.
Suitable for non-selective cultivation of E. coli strains for cloning, DNA plasmid production and production of recombinant proteins. Also suitable for selective cultivation when appropriate antibiotics are added.

[GUIDELINES]
Prepare enough for the necessary number of experiments to perform in order to prevent batch-to-batch variation between experiments. Input chemicals should be of a certain quality in order to prevent contaminants that can impact microbial growth and selection.

[STEPS]
SECTION: 1 000 mL 10x M9 salt solution
6. In a bottle, fill with 800 mL deionized water
SECTION: 1 000 mL 10x M9 salt solution
7. Measure and add:
    60 g Sodium phosphate (dibasic)
    30 g Monopotassium phosphate
    5 g Sodium chloride

Materials:
 
 
 
SECTION: 1 000 mL 10x M9 salt solution
8. Adjust pH to pH 7.4 using hydrogen chloride or sodium hydroxide
SECTION: 1 000 mL 10x M9 salt solution
9. Fill bottle to 900 mL with deionized water
SECTION: 1 000 mL 10x M9 salt solution
11. Autoclave solution at 121 °C for 45 min
SECTION: 1 000 mL 10x M9 salt solution
12. Aliquot into 100 mL units and store at 4 °C
SECTION: 100 mL 10x Ammonium chloride solution
1. In a bottle, fill with 400 mL deionized water
SECTION: 100 mL 1 M Magnesium sulfate solution
17. In a bottle, fill with 80 mL deionized water
SECTION: 100 mL 10x Ammonium chloride solution
4. Fill bottle to 500 mL with deionized water
SECTION: 100 mL 10x Ammonium chloride solution
2. Measure and add:
    50 g Ammonium chloride

Materials:
 
SECTION: 1 000 mL 10x M9 salt solution
10. Add 100 mL 10x Ammonium chloride solution
SECTION: 100 mL 10x Ammonium chloride solution
5. Sterilize with a 0.2-micron filter
SECTION: 100 mL 10x Ammonium chloride solution
3. Adjust pH to pH 7.4 using hydrogen chloride or sodium hydroxide
SECTION: 100 mL 1 M Magnesium sulfate solution
18. Measure and add:
    12.04 g Magnesium sulfate

Materials:
 
SECTION: 100 mL 0.5 M Calcium chloride solution
21. In a bottle, fill with 80 mL deionized water
SECTION: 500 mL M9 minimal medium
30. In a bottle, fill with 400 mL sterile water



SECTION: 100 mL 0.5 M Calcium chloride solution
24. Autoclave solution at 121 °C for 25 min
SECTION: 100 mL 0.5 M Calcium chloride solution
22. Measure and add:
    7.35 g Calcium chloride

Materials:
  
SECTION: 100 mL 1 M Magnesium sulfate solution
19. Fill bottle to 100 mL with deionized water
SECTION: 500 mL M9 minimal medium
32. Adjust pH to pH 7.4 using hydrogen chloride or sodium hydroxide
SECTION: 500 mL M9 minimal medium
31. Add:
    7.5 g Agar (Optional)
    50 mL 10x M9 salt solution
    10 mL Glucose solution (20%)
    1000 µL Magnesium sulfate solution (1 Molarity (M))
    100 µL Calcium chloride solution (0.5 Molarity (M))
    500 µL Thiamine hydrochloride solution (1 mg/mL)

 

Materials:
 
SECTION: 100 mL 1 M Magnesium sulfate solution
20. Autoclave solution at 121 °C for 25 min
SECTION: 500 mL 20% Glucose solution
13. In a bottle, fill with 400 mL deionized water
SECTION: 500 mL 20% Glucose solution
15. Fill bottle to 500 mL with deionized water
SECTION: 500 mL 20% Glucose solution
14. Measure and add:
    100 g Glucose

Materials:
 
SECTION: 100 mL 1 mg/mL Thiamine hydrochloride
25. In a bottle, fill with 80 mL deionized water
SECTION: 500 mL 20% Glucose solution
16. Autoclave solution at 121 °C for 40 min
SECTION: 100 mL 1 mg/mL Thiamine hydrochloride
27. Fill bottle to 100 mL with deionized water
SECTION: 100 mL 1 mg/mL Thiamine hydrochloride
26. Measure and add:
    100 mg Calcium chloride

Materials:
 

SECTION: 100 mL 0.5 M Calcium chloride solution
23. Fill bottle to 100 mL with deionized water
SECTION: 100 mL 1 mg/mL Thiamine hydrochloride
28. Sterilize with a 0.2-micron filter
SECTION: 100 mL 1 mg/mL Thiamine hydrochloride
29. Aliquot into 1 mL aliquots, and store at -20 °C
SECTION: 500 mL M9 minimal medium
33. Fill bottle to 500 mL with sterile water and store at 4 °C