Pattern preparation for LC/MS evaluation of depletion experiments
Rhizobium Leaf68 was cultivated in 20 ml tradition in a medium containing 20 amino acids and biotin, niacin, and pantothenate. In late-exponential section (OD~1), cells had been collected by way of centrifugation (10,000 rpm, 2 minutes), adopted by two rounds of washing and pelleting with 20 ml of sterile MgCl2 and 10,000 rpm for two min. Afterwards, cells had been collected by way of centrifugation and dissolved in 1 ml of MgCl2. Cultures had been inoculated in 4 shake flask cultures with 100 µl of this inoculum: one of many cultures was supplemented with all 3 nutritional vitamins, and a drop-out medium for every vitamin, respectively. OD was monitored as soon as per doubling, and the intracellular metabolome was sampled as follows. Cultures had been stored at 28 °C in a shaking water bathtub, and pattern quantity was decided primarily based on the OD (1/OD ml). The decided quantity of cell suspension was pipetted onto a filter standing atop a suction flask to take away the medium. Cells standing on filter had been washed with 10 ml of dH2O that was stored at 28 °C in water bathtub, and the washed filter was subsequently transferred to a Schott flask containing 8 ml of chilly acidified (−20 °C) quenching resolution (3 elements MeCN:1 half MeOH:1 half 0.05 M formic acid). After 10 minutes, the quenching resolution was transferred to a 50 ml Falcon tube and lyophilized at −50 °C in a single day. The ensuing powder was dissolved in 250 µl of pre-cooled dH2O, and stored in −80 °C till evaluation.
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