Fecal Wash Preparation

From Powers Wiki
Revision as of 18:49, 14 June 2022 by Mjeppesen (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


Metabolomics Sample Prep Protocol (Used for Rose project in February 2022)

  1. Centrifuge sample (14,000 xg, 10 min, 4º C) and remove supernatant to a clean tube (tube a)
  2. Dry supernatant overnight
  3. Wash pellet with 100% LCMS grade methanol
  4. Centrifuge and remove supernatant to a clean tube (tube b)
  5. Wash pellet with 50/50 LCMS grade methanol/water
  6. Centrifuge and remove supernatant to tube b
  7. Wash pellet with 100% LCMS grade water
  8. Centrifuge and remove supernatant to tube b
(Above three washes can be substituted for a number of 70:30 methanol:water washes)
  1. Dry all three fractions in speed vac
  2. Reconstitute samples into ~550 uL 7.0 pH adjusted potassum phosphate buffer (with TMSP) and vortex

Buffer Instructions

  • Make 50.0 mM Potassium Phosphate buffer at pH 7.0 in D2O
  • For 500.0 mL total solution, measure 2.177 g K2HPO4 (MW = 174.18 g/mol) and 1.70 g KH2PO4 (MW = 136.09g/mol) in separate 250.0 mL volumetric flasks and fill to line with 99.9% D2O.
  • Spike with 500 uL of TMSP from 10.0 uM stock solution