Triple Isotope (²H/¹³C/¹⁵N) M9 Growth Media

Autoclaved Materials

1. 2.8 L baffled flask

2. 1 L glass beaker with stir bar

3. 1 L glass bottle

4. 500 mL filtration apparatus (alternatively, use a 1 L “single use” sterile filter)

Media Preparation (1 L)

1. Add 750 mL D₂O to autoclaved beaker with stir bar.

2. Dissolve 6.85 g Na₂HPO₄ in 50 mL D₂O, and add to beaker.

3. Dissolve 3.05 g KH₂PO₄ in 20 mL D₂O, and add to beaker.

4. Dissolve 0.50 g NaCl in 10 mL D₂O, and add to beaker.

5. Dissolve 245 mg MgSO₄*7H₂O in 10 mL D₂O, and add to beaker.

6. Dissolve 2.0 g ¹⁵NH₄Cl in 10 mL D₂O, and add to beaker.

7. Dissolve 1.0 g ²H/¹³C/¹⁵N-isogrow in 50 mL D₂O, and add to beaker.

8. Dissolve 2.0 g ²H/¹³C-glucose in 40 mL D₂O, and add to beaker.

9. Add 10 mL 100x MEM vitamins to beaker.

10. Dissolve 80 mg CaCl₂ in 50 mL D₂O, and add to beaker.
    1. The dilution of CaCl₂ prior addition to the beaker solution is important to prevent the precipitation of calcium phosphate.
11. Add desired antibiotic.
    1. e.g. 100 mg ampicillin or 50 mg kanamycin.

12. OPTIONAL Add 5 mg thiamine HCl (Vitamin B₁)

13. Cover beaker with aluminum foil and let stir ~20 min.

14. Pass media through 0.22 μm filter using steriled filration apparatus into autoclaved 1 L glass bottle.

15. Transfer media to 2.8 L baffled flask near open flame, and cover flask with aluminum foil.