Chapter 6 Soil Extractions

6.1 Detailed Procedure from DNeasy Powersoil Extraction kit

  1. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 mg of soil and 800 µl of Solution CD1. Vortex briefly to mix.

    Note: PowerBead Pro Tube contains a buffer that will help disperse the soil particles, begin to dissolve humic acids, and protect nucleic acids from degradation.

  2. Homogenize samples thoroughly - Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5–2 ml tubes. Vortex at maximum speed for 10 min.

    Note: If using the Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5–10 min.

  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. The supernatant may still contain some soil particles.

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

    Note: Solution CD2 contains IRT, which will remove contaminating organic and inorganic matter that may reduce DNA purity.

  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.

    Note: Expect 500–600 µl.

  7. Add 600 µl of Solution CD3 and vortex for 5 s.

    Note: Solution CD3 will adjust the DNA solution salt concentration to allow binding of DNA to the MB Spin Column filter membrane.

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

    Note: DNA is selectively bound to the silica membrane in the MB Spin Column.

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

  10. Carefully place the MB Spin Column into a clean 2 ml Collection Tube. Avoid splashing any flow-through onto the MB Spin Column.

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

    Note: Solution EA is a wash buffer and is removing protein and other non-aqueous contaminants.

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

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

    Note: Solution C5 is an ethanol-based wash solution to further clean the DNA.

  14. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube.

  15. Centrifuge at up to 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube.

    Note: This spin removes residual Solution C5, which is critical.

  16. Add 50–100 µl of Solution C6 to the center of the white filter membrane.

    Note: Releases DNA from the silica membrane.

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

    Note: Store the DNA frozen (–30 to –15°C or –90 to –65°C) as Solution C6 does not contain EDTA.