Mar 20, 2023
Standard M9 minimal medium
This protocol is a draft, published without a DOI.
- 1University of Oslo
Protocol Citation: Andreas Sagen 2023. Standard M9 minimal medium. protocols.io https://protocols.io/view/standard-m9-minimal-medium-crd7v29n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 17, 2023
Last Modified: March 20, 2023
Protocol Integer ID: 79007
Keywords: M9 minimal medium, e. coli, Escherichia coli, bacteria, autotrophic medium
Abstract
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.
Materials
Magnetic stirrer, autoclave, scale, tubes, flask
Protocol materials
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
Step 7
Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0876
Step 7
Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5379
Step 7
Ammonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4514
Step 2
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
Step 18
Calcium chloride dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3881
Step 22
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Step 31
GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
Step 14
Thiamine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1270
Step 26
Safety warnings
Be sure to wear appropriate PPE when working with antibiotics and chemicals. Take care when working with hot flasks and tubes.
100 mL 10x Ammonium chloride solution
100 mL 10x Ammonium chloride solution
1h
1h
In a bottle, fill with 400 mL deionized water
5m
Measure and add:
50 g Ammonium chloride
Materials:
Ammonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4514
15m
Adjust pH to 7.4 using hydrogen chloride or sodium hydroxide
5m
Fill bottle to 500 mL with deionized water
2m
Sterilize with a 0.2-micron filter
20m
1 000 mL 10x M9 salt solution
1 000 mL 10x M9 salt solution
2h
2h
In a bottle, fill with 800 mL deionized water
5m
Measure and add:
60 g Sodium phosphate (dibasic)
30 g Monopotassium phosphate
5 g Sodium chloride
Materials:
Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0876
Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5379
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
15m
Adjust pH to 7.4 using hydrogen chloride or sodium hydroxide
5m
Fill bottle to 900 mL with deionized water
2m
Add 100 mL 10x Ammonium chloride solution
1m
Autoclave solution at 121 °C for 00:45:00
45m
Aliquot into 100 mL units and store at 4 °C
30m
500 mL 20% Glucose solution
500 mL 20% Glucose solution
1h 30m
1h 30m
In a bottle, fill with 400 mL deionized water
5m
Measure and add:
100 g Glucose
Materials:
GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
15m
Fill bottle to 500 mL with deionized water
5m
Autoclave solution at 121 °C for 00:40:00
40m
100 mL 1 M Magnesium sulfate solution
100 mL 1 M Magnesium sulfate solution
45m
45m
In a bottle, fill with 80 mL deionized water
2m
Measure and add:
12.04 g Magnesium sulfate
Materials:
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
5m
Fill bottle to 100 mL with deionized water
1m
Autoclave solution at 121 °C for 00:25:00
25m
100 mL 0.5 M Calcium chloride solution
100 mL 0.5 M Calcium chloride solution
45m
45m
In a bottle, fill with 80 mL deionized water
2m
Measure and add:
7.35 g Calcium chloride
Materials:
Calcium chloride dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3881
10m
Fill bottle to 100 mL with deionized water
1m
Autoclave solution at 121 °C for 00:25:00
25m
100 mL 1 mg/mL Thiamine hydrochloride
100 mL 1 mg/mL Thiamine hydrochloride
45m
45m
In a bottle, fill with 80 mL deionized water
2m
Measure and add:
100 mg Calcium chloride
Materials:
Thiamine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1270
10m
Fill bottle to 100 mL with deionized water
1m
Sterilize with a 0.2-micron filter
2m
Aliquot into 1 mL aliquots, and store at -20 °C
30m
500 mL M9 minimal medium
500 mL M9 minimal medium
20m
20m
In a bottle, fill with 400 mL sterile water
5m
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 )
Note
If using agar, autoclave solution with agar only, then cool to approx. 50 °C , then add the other components. Keep hot enough for the medium to stay liquid, then pour immediately.
Materials:
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
5m
Adjust pH to 7.4 using hydrogen chloride or sodium hydroxide
5m
Fill bottle to 500 mL with sterile water and store at 4 °C
Note
While it shouldn't be necessary to sterilize if working aseptically, it is possible to sterilize using a 0.2-micron filter. DO NOT AUTOCLAVE!
Note
Supplement medium with antibiotics, etc. as necessary
5m