Metabolite extraction from E.coli

1.      Inoculate E.coli cells from O/N grown culture in 3ml LB at 0.01 OD

2.      Grow for 5 hrs at 37ᴼC, 200rpm or till OD reaches 1

3.      Take cells equivalent to 0.1 OD and proceed with extraction

4.      Pellet cells at 14000rpm, 1min, 4ᴼC

5.      Discard supernatant and add 200ul 80% chilled methanol (80% MetOH, PIPES 1ug/ul, C13 labeled glutamine)

6.      Vortex briefly and sonicate in water bath for 15min at 4ᴼC with intermittent vortexing every 5mins

7.      Spin at 14000rpm, 5min, 4ᴼC

8.      Collect supernatant in glass vial and add 100ul 80% chilled methanol to the pellet

9.      Sonicate in water bath for 5min at 4ᴼC with intermittent vortexing every 2mins

10.  Spin at 14000rpm, 5min, 4ᴼC

11.  Collect supernatant in the previous glass vial and add 100ul 80% chilled methanol to the pellet

12.  Sonicate in water bath for 5min at 4ᴼC with intermittent vortexing every 2mins

13.  Spin at 14000rpm, 5min, 4ᴼC

14.  Collect supernatant in the same glass vial.

5 injections done for each sample (5 biological replicates)