Fecal Wash Preparation: Difference between revisions
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Metabolomics Sample Prep Protocol (Used for Rose project in February 2022) | |||
# Centrifuge sample (14,000 xg, 10 min, 4º C) and remove supernatant to a clean tube (tube a) | |||
# Dry supernatant overnight | |||
# Wash pellet with 100% LCMS grade methanol | |||
# Centrifuge and remove supernatant to a clean tube (tube b) | |||
# Wash pellet with 50/50 LCMS grade methanol/water | |||
# Centrifuge and remove supernatant to tube b | |||
# Wash pellet with 100% LCMS grade water | |||
# Centrifuge and remove supernatant to tube b | |||
:: (Above three washes can be substituted for a number of 70:30 methanol:water washes) | |||
# Dry all three fractions in speed vac | |||
# 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 | |||
Latest revision as of 18:49, 14 June 2022
Metabolomics Sample Prep Protocol (Used for Rose project in February 2022)
- Centrifuge sample (14,000 xg, 10 min, 4º C) and remove supernatant to a clean tube (tube a)
- Dry supernatant overnight
- Wash pellet with 100% LCMS grade methanol
- Centrifuge and remove supernatant to a clean tube (tube b)
- Wash pellet with 50/50 LCMS grade methanol/water
- Centrifuge and remove supernatant to tube b
- Wash pellet with 100% LCMS grade water
- Centrifuge and remove supernatant to tube b
- (Above three washes can be substituted for a number of 70:30 methanol:water washes)
- Dry all three fractions in speed vac
- 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