Additional Extraction Protocols

Corrinoid Extraction

1. Grow cells in corrinoid free media
2. Pellet down cells (20mL of 10^9 cell density)
3. Resuspend cells in 10mL of MilliQ water containing 10mM KCN and boil for 30min.
4. Centrifuge at 16000 x g for 20min
5. Apply supernatant to washed and equilibrated Sep-Pak C18 cartridge (0.5g).
6. Wash with 10mL of water and 5mL of 10% MeOH.
7. Elute with 5mL of 70% MeOH.
8. Concentrate sample under vacuum.
9. Resuspend in 200µL MilliQ water

Total Amino Acid Extraction (Not Suitable for Tryptophan)

1. Lyophilize sample.
2. Grind sample to fine powder (capable of passing through 0.5mm sieve).
3. Weigh 0.15g dry material into falcon tube and cool to 0°C.
4. Add 1mL of oxidation solution, seal falcon tube, mix and leave at 0°C for 16hrs.
5. Add 186mg sodium metabisulfite (decompose excess oxidation reagent).
6. Add 5mL hydrolysis solution to sample.
7. Place unsealed falcon tube in oven at 110°C for 1hr.
8. Seal falcon tube and leave in oven for 24hrs.
9. Remove falcon tube from oven and carefully open.
10. Place sample in ice bath and allow to cool.
11. Filter with 10kDa filter.

Formic Acid-Phenol Solution: 889g formic acid + 111 mL water + 4.73g phenol
Oxidation Solution: 0.5mL 30% hydrogen peroxide + 4.5mL formic acid-phenol solution (mix for 1hr at room temperature then cool 15min in ice bath)
Hydrolysis Solution: 1g phenol in 1L of 6N HCl

Free Fatty Acid Extraction

1. Lyophilize sample.
2. Grind sample to fine powder (capable of passing through 0.5mm sieve).
3. Weigh 0.05g into a 2mL Eppendorf tube.
4. Add 5 metal balls (2mm diameter) and 1.5mL of EtOH
5. Vortex/bead beat samples (10 times 10 sec pulses, cool on ice in between pulses).
6. Centrifuge at max speed in microcentrifuge for 5min.
7. Filter 0.6mL supernatant using 10kDa filters
8. Dry samples in speedvac.

Fat Soluble Vitamin Extraction

1. Lyophilize sample.
2. Grind sample to fine powder (capable of passing through 0.5mm sieve).
3. Weigh 0.2g into a 2mL Eppendorf tube.
4. Add 5 metal balls (2mm diameter) and 1.5mL of EtOH
5. Vortex/bead beat samples (10 times 10 sec pulses, cool on ice in between pulses).
6. Centrifuge at max speed in microcentrifuge for 5min.
7. Filter 0.6mL supernatant using 10kDa filters
8. Dry samples in speedvac.

Water Soluble Vitamin Extraction (Not Suitable for Vitamin C)

1. Lyophilize sample.
2. Grind sample to fine powder (capable of passing through 0.5mm sieve).
3. Weigh 0.05g into a 2mL Eppendorf tube.
4. Add 5 metal balls (2mm diameter) and 1mL of 1% ascorbic acid
5. Vortex/bead beat samples (10 times 10 sec pulses, cool on ice in between pulses).
6. Neutralize with 3.2µL of concentrated Ammonium hydroxide solution.
7. Centrifuge at max speed in microcentrifuge for 5min.
8. Filter 0.6mL supernatant using 10kDa filters
9. Dry samples in speedvac.