The following is a protocol for growing 1.0 L of E. coli in deuterium oxide (D2O)-M9 salts medium. Due to the slow growth of cells in D2O minimal medium, the D2O culture is inoculated with cells at 0.3 A600 grown on H2O -M9 Salts medium.

To prepare 1L of D2O medium, follow all the 11 steps listed in the Minimal Salts Media protocol, but a) substitute the water with deuterium oxide (D2O) in all the solutions prepared, and b) do not autoclave D2O itself, MgSO4, glucose, CaCl2, or yeast extract for sterilization, only filter (0.22mM filter). Also if using 15N or 13C or both, remember to prepare the 15NH4Cl or 13C-glucose, respectively in D2O as well.

The protocol below is for culturing cells on 1.0 L of D2O medium starting from cells growing on H2O-M9 salts medium:

1. Inoculate 200mL of H2O-M9 salts medium from fresh colony (as described in Minimal Salts media protocol)

2. Let cells rise to an A600 of ~0.3.

3. Centrifuge initial 200mL cells in sterile bottles and dump supernatant.

4. Resuspend pellet in 10mL of D2O-M9 medium (prepared with 15N or 13C isotope as appropriate).

5. Add the 10mL of resuspended cells to the 1.0 L D2O culture in sterile flask. This will yield a starting A600 of ~ 0.06.

6. Wait for cells to rise at an A600 of ~0.7, then induce protein expression with IPTG (or whatever is appropriate) and add 2.5mL of 20% (w/v) D-glucose (or isotope labeled 13C-glucose, as appropriate).

7. After induction is complete (typically 5-7 hours), spin down rest of cells 10 minutes at 7500 r.p.m.