Magni:Protocols/M9

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M9 supplemented medium (liquid)

The final composition of the medium is: M9 salts, 1 mM thiamine hydrochloride, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glycerol.

• For every 1 L of medium, add to a bottle or flask (in the following order) while using a sterile magnetic stirrer:
  • 739 ml of autoclaved deionized water (blue tap of the Millipore machine)
  • 100 ul of CaCl2 1M
  • 200 ml of 5X M9 salts
  • 20 ml of casamino acids 10%
  • 34 ml of thiamine hydrochloride 10 mg/ml
  • 5 ml of glycerol 80%
  • 2 ml of MgSO4 1M
• Store in the fridge. NOTE: medium occasionally precipitates when stored for long periods.

CaCl2 1M

• Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
• Bring to a volume of 100 ml.
• Autoclave in a pyrex bottle.
• Store in the fridge and use at room temperature, near the flame.

5X M9 salts

• Dissolve 56.4 g of powder in approximately 800 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
• Bring to a volume of 1 L.
• Autoclave in a pyrex bottle.
• Store in the fridge and use at room temperature, near the flame.

Casamino acids 10%

• Dissolve 10 g of powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer (the solution will not become clear before autoclaving).
• Bring to a volume of 100 ml.
• Autoclave in a pyrex bottle.
• Store in the fridge and use near the flame.

Thiamine hydrochloride 10 mg/ml

• Dissolve 0.4 g of powder in exactly 40 ml of Milli-Q water (from the filter of the Millipore machine) in a 50-ml tube.
• Filter-sterilize with a 20-ml syringe and a 0.2 um filter.
• Do not store! use it when needed to prepare the medium.

Glycerol 80%

• Mix 80 ml of glycerol with 20 ml of deionized water (blue tap of the Millipore machine).
• Autoclave in a pyrex bottle.
• Store at room temperature and use near the flame.

MgSO4 1M

• Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
• Bring to a volume of 100 ml.
• Autoclave in a pyrex bottle.
• Store in the fridge and use at room temperature, near the flame.