Chapter 4 Poop Extractions

4.1 Fecal Pellet Storage:

  • When a fecal pellet is released, it should be put in a 2 mL tube with a 20% glycerol solution. The volume of glycerol solution added depends on the size of the pellet: it should be enough to cover the entire pellet but shouldn’t dilute it too much.

  • Store at -20 ℃

4.1.1 Notes Before Beginning:

-Solution CD2 should be stored at 2-8℃ , all other reagents should be stored at room temperature (15-25℃).

-Heat solution CD3 and C6 to 65 ℃ in the water bath.

-Secure the fecal samples in the glycerol solutions on a Vortex Adapter and vortex at maximum speed for 10 minutes.

4.2 DNA Extraction Protocol:

Protocol modified by the Tabima lab (2022) from the Qiagen DNeasy PowerSoil Pro Kit (2019).

  1. Spin the Powerbead Pro Tube briefly to ensure that the beads have settled to the bottom. Add 200-300 µL of homogenized fecal sample and 800 µL of Solution CD1. Vortex briefly to mix.

  2. Secure the PowerBead Pro Tube horizontally on the Vortex Adapter and vortex at maximum speed for 10-15 minutes.

  3. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.

  4. Transfer the supernatant to a clean 2 mL Microcentrifuge tube (Note: Expect 500-600 µl).

  5. Add 200 µl of Solution CD2 and vortex from 5 s.

  6. Centrifuge at 15,000 x g for 1 min at room temperature. Avoiding the pellet, transfer up to 700 µL of supernatant to a clean 2 mL Microcentrifuge tube.

7.Add 600 µL of solution CD3 and vortex for 5 s.

  1. Load 650 µL of the lysate onto an MB Spin Column and centrifuge at 15,000 x g for 1 min.

  2. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.

  3. Carefully place the MB Spin Column into a clean 2 mL collection tube. Avoid splashing any flow-through onto the MB Spin Column.

  4. Add 500 µL of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.

  5. Discard the flow-through and place the MB Spin Column back into the same 2 mL Collection Tube.

  6. Add 500 µL of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.

  7. Discard the flow-through and place the MB Spin Column into a new 2 mL collection tube.

  8. Centrifuge at 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 mL Elution Tube.

  9. Add 50-100 µL of Solution C6 to the center of the white filter membrane.

  10. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.

18.Store DNA at -20 ℃ .