M9 minimal medium
Isaac Sir Núñez, Tamara Matute
Abstract
M9 minimal medium is a classical bacterial growth broth (Miller, et al., 1972) characterized by its low autofluorescence.
As it is minimal, it can be supplemented with defined carbon sources or amino acids acord the custom experimental setups.
Steps
Recipe
This is the recipe for 500 mL total medium.
It is assembled from sterile reagents in sterile conditions (in a laminar flow or flame, and using a sterille glass bottle). They have to be added in this order and mixed well after incopore each component.
250mL Sterile Molecular grade water
100mL 5X M9 Salt Mix
1mL MgSO4:7H2O (1M)
10mL Glucose (20% w/v) --> 0.4 w/v final concentration in the medium
50µL CaCl2 (1M)
100mL Casaminoacids (1%)
complete with ~39mL Sterile Molecular grade water
In case of solid media preparation:
39mL Sterile Molecular grade water
100mL 5X M9 Salt Mix
1mL MgSO4:7H2O (1M)
10mL Glucose (20% w/v) --> 0.4 w/v final concentration in the medium
50µL CaCl2 (1M)
100mL Casaminoacids (1%)
melt Agar 3% in a microwave or hot bath and complete the media with 250mL of it.
It should be added quite hot to allow you mix it well and give you enough time to do the plates before it gets solid.
Antibiotic supplementation can be done after adding the Agar.
Plates could be stores at 4°C and darkness for a couple of days but sometimes they form precipitate.
*** You can replace the carbon source or aminoacids for any other(s) accord your experimental setup ***
Reagents preparation
5X M9 Salts (600 mL)
Weight these amounts of each salt and add to a glass bottle:
38.4g Na2HPO4:7H2O
9g KH2PO4
1.5g NaCl
3g NH4Cl
Then, add 600mL Molecular grade or deionized water and dissolve completely.
Sterilize by autoclaving
MgSO4:7H2O (1M) - 20 mL 4:7H2O (1M) - 20 mL
Weight 4.930g of MgSO4:7H2O
Complete to 20mL with Molecular grade or deionized water and dissolve completely.
Sterilize by autoclaving
Glucose (20% w/v) - 100 mL
Weight 20g of Glucose.
Complete to 100mL with Molecular grade or deionized water and dissolve completely.
Sterilize by filtering with 0.22 µm syringe filter.
CaCl2 (1M) - 5 mL 2 (1M) - 5 mL
Weight 0.555g of CaCl2.
Complete to 5mL with Molecular grade or deionized water and dissolve completely.
Sterilize by filtering with 0.22 µm syringe filter.
Casaminoacids (1%) - 100 mL
Weight 1g of Casaminoacids.
Complete to 100mL with Molecular grade or deionized water and dissolve completely.
Sterilize by autoclaving.
Agar 3% - 500 mL
Weight 15g of Agar (molecular biology grade).
Complete to 500mL with deionized water and dissolve completely.
Sterilize by autoclaving.
