DNeasy PowerSoil Protocol

Before you Begin:

Note: Upon arrival of the kit the CD2 solution should be stored at 2–8°C upon arrival. All other reagents and kit components should be stored at room temperature (15-25°C).

Material:

• Quick-Start Protocol for the DNeasy PowerSoil Pro Kit as published in the May 2019 DNeasy® PowerSoil® Pro Kit Guide as seen here www.qiagen.com/HB-2495. The safety data sheet can be found here: www.qiagen.com/safety.

• Before starting ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing. If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves. Perform all centrifugation steps at room temperature (15-25°C).

Protocol:

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 ul of Solution CD1. Vortex briefly to mix.

2. Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5-2 ml tubes (cat. no. 13000-V1-24). 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.

a. For more information about other bead beating methods, see the “Protocol: Detailed" section of DNeasy® Power Soil® Pro Kit Handbook.

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 (provided).

a. Expect 500-600 ul. The supernatant may still contain some soil particles.

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

6. Centrifuge at 15,000 xg for 1 min at room temperature. Avoiding the pellet, transfer up to 700 ul of supernatant to a clean 2 ml Microcentrifuge Tube (provided).

a. Expect 500-600 ul.

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

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

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 (provided). Avoid splashing any flow-through onto the MB Spin Column.

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

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

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

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

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 (provided).

16. Add 50-100 ul of Solution C6 to the center of the white filter 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: We recommend storing the DNA frozen (-30 to -15°C or -90 to -65°C) as Solution Co does not contain EDTA. To concentrate DNA, please refer to the Troubleshooting Guide.