Protocol 1 | Protocol 2 | Protocol 3 | Protocol 4 | Protocol 5 | Protocol 6 | Protocol 7 | Protocol 8 |
50 mg stool | 50 mg stool | 50 mg stool | 50 mg stool | 50 mg stool | 50 mg stool | 50 mg stool | 50 mg stool |
150 µL PBS | 200 µL isopropanol | 200 µL Methanol/ acetonitril/ H2O * (2/2/1) | 200 µL Methanol/ acetonitril (1/1) | 150 µl 85% Ethanol/15% 20 mM phosphate buffer | 200 µL 75% Ethanol | 225 µL 100% Methanol | 150 µL 100% methanol |
Vortex 2 min | |||||||
Freeze in liquid N2 (1 min); thaw | |||||||
5 min sonification on ice | |||||||
500 µL MTBE | 750 µL MTBE | ||||||
Shake 1 hour at RT | Shake 1 hour at 4°C | ||||||
188 µL H2O * + 0.1% ammonium acetate | |||||||
Vortex 2 min, incubate 5 min at RT | |||||||
Centrifugation at full speed (5 min) | Centrifugation at full speed (10 min) | Centrifugation at full speed (5 min) | |||||
Supernatant | Upper supernatant | Supernatant | |||||
150 µL 20% Ethanol/80% 20 mM phosphate buffer | 125 µL H2O * | ~2000 µL 100% methanol | 150 µL 20% methanol | ||||
Vortex 2 min | Vortex 1 min | ||||||
Incubate 10 min at RT | Incubate 1 hour at −20°C | ||||||
Centrifugation at full speed at 4°C (15 min) + supernatant in extra tube | |||||||
Freeze in liquid nitrogen | Freeze in liquid nitrogen | Freeze in liquid nitrogen | Freeze in liquid nitrogen | Freeze in liquid nitrogen | Dry complete in SpeedVac | Dry complete in SpeedVac | Freeze in liquid nitrogen |
*DNAse-free and RNAse-free water.
MTBE, methyl tert-butyl ether; PBS, phosphate-buffered saline; RT, room temperature.