Protocol for extracting ppGpp from amino acids or carbon starved cells using AA or Glucose analogs

1. Grow 2-3 ml of cells in minimal T-salts medium supplemented with 0.2 % glucose and 1 mM KH₂PO₄
2. At the exponential growth phase (OD₆₀₀ ≈ 0.5), wash with T-buffer.  Resuspend in the same volume of T-salts supplemented with 0.1% glucose
   and 0.25 mM Pi
3. Measure OD and dilute cells to a final OD₆₀₀=0.1- 0.15 T-salts supplemented with 0.1% glucose and 0.25 mM Pi (use the lowest possible volume, usually 200 μl is sufficient). Add ³²Pi to a final concentration of 100 μCi/ml and let cells grow for at least one hour
4. Take a 20 μl sample and mix it with 10 μl of 11 M cold formic acid for the basal ppGpp level. Keep the extract on ice until the end of the experiment
5. Split the culture into 2 portions of 0.2 ml each. To one portion add α-methyl glucoside to a final concentration of 2% and to the other serine hydroxamate to a concentration of 1 mg/ml
6. Take 20 μl samples at 0, 5 and 15 min following α-methyl glucoside or serine hydroxamate addition and mix with 20 μl 11 M cold formic acid. Keep the extracts on ice
7. Freeze the samples at -70^oC, thaw on ice
8. Apply 2-5 μl of the formic acid extrats to PEI plates and resolve nucleotides in 1.5 M KH₂PO₄ pH 3.4.