TITLE: QIAGEN® DNeasy® PowerSoil® Pro
AUTHORS: Hsin-Mao  Wu

[DESCRIPTION]
For the isolation of microbial genomic DNA from all soil types, including difficult samples such as compost, sediment, and manure.

[BEFORE_START]
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).

[STEPS]
SECTION: Prepare sample &amp; Cell lysis
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.
 

SECTION: Prepare sample &amp; Cell lysis
2. Homogenize samples thoroughly using one of the following methods:
SECTION: Inhibitor removal
5. Add 200 µL  of Solution CD2 and vortex for 5 s .
 


SECTION: Prepare sample &amp; Cell lysis
2.1. 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 .
 

 


SECTION: Prepare sample &amp; Cell lysis
2.2. Use a PowerLyzer 24 Homogenizer. PowerBead Pro Tubes must be properly balanced in the tube holder of the PowerLyzer 24 Homogenizer. We recommend homogenizing the soil at 2000 rpm, 30 s , pausing for 30 s , then homogenizing again at 2000 rpm, 30 s .

 

SECTION: Prepare sample &amp; Cell lysis
2.3. Use a TissueLyser II. Place the PowerBead Pro Tube into the TissueLyser Adapter Set  2 x 24 (cat. no. 69982) or 2 ml Tube Holder (cat. no. 11993) and Plate Adapter  Set (cat. no. 11990). Fasten the adapter into the instrument and shake for5 min  at  speed 25 Hz. Reorient the adapter so that the side that was closest to the machine body is now furthest from it. Shake again for 5 min at a speed of 25 Hz.

 

SECTION: Prepare sample &amp; Cell lysis
3. Centrifuge the PowerBead Pro Tube at 15000 rpm, 1 min .

SECTION: Prepare sample &amp; Cell lysis
4. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided).
 

SECTION: Inhibitor removal
6. Centrifuge at 15000 x g, 1 min . Avoiding the pellet, transfer up to 700 µL  of supernatant to a clean 2 ml Microcentrifuge Tube (provided).
 


 

SECTION: Bind DNA
7. Add 600 µL  of Solution CD3 and vortex for 5 s .
 


SECTION: Bind DNA
8. Load 650 µL  of the lysate onto an MB Spin Column and centrifuge at 15000 x g, 1 min . 
 


SECTION: Bind DNA
9. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
SECTION: Bind DNA
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.
SECTION: Wash
11. .Add 500 µl of Solution EA to the MB Spin Column. Centrifuge at 15000 x g, 1 min .
 


SECTION: Wash
12. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
SECTION: Wash
13. Add 500 µL  of Solution C5 to the MB Spin Column. Centrifuge at 15000 x g, 1 min .
 


SECTION: Wash
14. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).
SECTION: Wash
15. Centrifuge at up to 16000 x g, 2 min . Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided).
 


SECTION: Elute
16. Add 50 µL  ~100 µL of Solution C6 to the center of the white filter membrane.
 


SECTION: Elute
17. Centrifuge at 15000 x g, 1 min . Discard the MB Spin Column. The DNA is now ready for downstream applications.