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Metabolomics Sample Preparation
Author: Tyler Hilsabeck, updated date: , view: 18, Q&A: 0
Tag: Metabolomics

Equipment

  1. Diagenode sonicator

  2. Vortex

  3. Centrifuge

  4. Micropipette

Procedure

  1. Collect 5 eppendorf tubes with 5 whole flies per tube (25, total), calculating mass per fly for each set of 5.
    Store at -80C

  2. Add 15 uL of Fly Solvent per fly to each tube.
    Keep samples in dry ice.

  3. Turn on Diagenode and allow it to cool to 4C.

  4. Sonicate first set of 6 samples for 10 cycles at 30 seconds/cycle, then transfer to dry ice.

  5. While the next set of 6 samples is sonicating, briefly vortex the previous set of samples to break up and disperse remaining intact fly parts. Make sure all large portions are in the solution, briefly centrifuge if necessary.
    If tasks are split between two people, the person sonicating should prepare labels for HPLC vials.

  6. Sonicate each set of 6 samples a second time for 5 cycles of 30 seconds/cycle.

  7. After the second round of sonication, alternate vortexing and letting the samples sit in dry ice until all the large fly parts are broken apart. Allow the samples to sit in dry ice for ~3 minutes each time, giving the methanol time to break down the fly innards. Do this until all large fly portions are broken down (usually 1 or 2 repetitions).

  8. Let samples sit at -20C for 1 hour, to precipitate out the protein.

  9. Vortex samples.

  10. Centrifuge samples for 7-10 min at 15K rpm.

  11. Collect supernatant into labeled HPLC vials, store at -20C if analyzing soon, -80C, otherwise.

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