Knight:M9 supplemented media

This is our protocol for making supplemented M9 medium.

Component Concentrations

• 1X M9 salts
• 1mM thiamine hydrochloride
• 0.4% glycerol
• 0.2% casamino acids
• 2mM MgSO₄
• 0.1mM CaCl₂

Protocol

For 1L of 1X media

• 200 mL 5X M9 minimal salts

1. Dissolve 56.4 g Bacto M9 minimal salts, 5X from Difco in 1L H₂O
2. Separate into 200 mL aliquots
3. Autoclave to sterilize. 121°C for 15 minutes.

• 34 mL 10 mg/mL thiamine

1. Dissolve 10 mg per mL of H₂O
2. Use a 0.22 μm filter to filter-sterilize

• 10 mL 40% glycerol

1. Add 80 mL glyerol to 120 mL of H₂O
2. Autoclave to sterilize

• 20 mL 10% Casamino acids

1. Dissolve 50 g Bacto Casamino acids from Difco in 500 mL H₂O
2. Autoclave to sterilize

• 2 mL 1M MgSO₄

1. Dissolve 24.65 g MgSO₄·7H₂O in 100 mL H₂O
2. Autoclave to sterilize

• 100 μL 1M CaCl₂

1. Dissolve 14.7 g CaCl₂·2H₂O in 100 mL H₂O
2. Autoclave to sterilize

• 733.9 mL H₂O

1. Sterilize deionized water in autoclave

Combine above solutions using sterile technique. (May notice some precipitation during preparation but precipitate should go back into solution once volume is brought up to 1L with sterile H₂O.)
Add antibiotic as appropriate and store at 4°C