Triple Isotope (2H/13C/15N) M9 Growth Media
Autoclaved Materials
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2.8 L baffled flask
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1 L glass beaker with stir bar
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1 L glass bottle
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500 mL filtration apparatus (alternatively, use a 1 L “single use” sterile filter)
Media Preparation (1 L)
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Add 750 mL D2O to autoclaved beaker with stir bar.
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Dissolve 6.85 g Na2HPO4 in 50 mL D2O, and add to beaker.
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Dissolve 3.05 g KH2PO4 in 20 mL D2O, and add to beaker.
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Dissolve 0.50 g NaCl in 10 mL D2O, and add to beaker.
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Dissolve 245 mg MgSO4*7H2O in 10 mL D2O, and add to beaker.
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Dissolve 2.0 g 15NH4Cl in 10 mL D2O, and add to beaker.
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Dissolve 1.0 g 2H/13C/15N-isogrow in 50 mL D2O, and add to beaker.
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Dissolve 2.0 g 2H/13C-glucose in 40 mL D2O, and add to beaker.
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Add 10 mL 100x MEM vitamins to beaker.
- Dissolve 80 mg CaCl2 in 50 mL D2O, and add to beaker.
- The dilution of CaCl2 prior addition to the beaker solution is important to prevent the precipitation of calcium phosphate.
- Add desired antibiotic.
- e.g. 100 mg ampicillin or 50 mg kanamycin.
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OPTIONAL Add 5 mg thiamine HCl (Vitamin B1)
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Cover beaker with aluminum foil and let stir ~20 min.
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Pass media through 0.22 μm filter using steriled filration apparatus into autoclaved 1 L glass bottle.
- Transfer media to 2.8 L baffled flask near open flame, and cover flask with aluminum foil.