Preparation of bacterial samples
1. Starter bacterial culture is grown overnight (optimal temperature/medium).
2. Inoculate sample cultures (20mL culture per data point).
3. Grow to culture to desired cell density.
4. Rapidly isolate cells from culture media (to avoid starvation and subsequent degradation of metabolites) using either a vacuum manifold equipped with a 0.45µm nylon membrane, or by centrifugation.
5. Immediately add isolated cells to 1.5mL of -20°C extraction solution in 15mL falcon tube, vortex and place at -20degC for 30min.
6. Transfer extraction solution to 2mL eppendorf and spin at max speed, 4°C for 5min.
7. Transfer supernatant to new 2mL eppendorf (store at -20°C), resuspend pellet in 300uL of extraction solution, place at -20°C for 30min then spin at max speed, 4°C for 5min.
8. Add supernatant to previous 2mL eppendorf (store at -20°C), resuspend pellet in 200uL of extraction solution, place at -20°C for 30min then spin at max speed, 4°C for 5min.
9. Add supernatant to previous 2mL eppendorf, add 7uL Ammonium hydroxide per mL of extraction solution and mix thoroughly (to neutralize).
10. Split into two equal fractions and store at -80°C overnight.
11. Concentrate each sample under vacuum and re-suspend in 100uL of H2O.
Extraction solution: 0.1M formic acid in MeOH:Acetonitrile:H2O (40:40:20)
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