Soil DNA Extraction — DNeasy 96 PowerSoil Pro

Bench manual for 96-well plate soil DNA extraction using the QIAGEN DNeasy 96 PowerSoil Pro Kit.

Format96-well plate

Input≤0.25 g soil/well

Elution volume100 µL

Elapsed time~2–3 h

Before Starting

Storage

Solution CD2 must be stored at 2–8°C upon arrival. All other reagents store at room temperature (15–25°C).

Notes

If Solution CD3 has precipitate, heat at 60°C until dissolved before use.
Use a 96-well plate rotor for all centrifugation steps; perform at room temperature.
Use extra-long pipette tips (1000–1250 µL) for collection microtubes (CMTRs).

Kit Solutions

Kit Hardware

Equipment

TissueLyser II with Adapter Plate Sets (cat. no. 11990) and silicone compression mat
Centrifuge with 96-well plate rotor, capable of 4500 × g
Vortex mixer

~30–40 min

Briefly centrifuge the PowerBead Pro Plate to settle the beads at the bottom of each well.
Remove and discard the square well mat. Add ≤0.25 g soil (or ≤0.1 g stool) and 800 µL Solution CD1 to each well.
Remove any residual liquid from the top of the plate. Seal firmly with the sealing film provided. A mechanical plate sealer gives the most consistent seal.
Place silicone compression mat on top of the sealing film. Sandwich the plate and mat between 2 Adapter Plate Sets. Shake at 25 Hz for 5 min.
Re-orient the plate so the side closest to the machine body is now furthest. Shake again at 25 Hz for 5 min. Do not exceed 2 × 5 min at 25 Hz.
Centrifuge the PowerBead Pro Plate at room temperature for 6 min at 4500 × g.
Discard the sealing film. Transfer supernatant to collection microtubes (CMTRs). Expect 500–600 µL; some soil particles may remain.

~20–25 min

Add 200 µL Solution CD2 to each CMTR. For very high-inhibitor samples, use 250 µL. Seal with caps provided and vortex.
Centrifuge at room temperature for 6 min at 4500 × g.
Transfer up to 700 µL supernatant to an S-Block. Expect 500–600 µL.
Add 600 µL Solution CD3 to each well of the S-Block. Pipette up and down to mix. Place a QIAamp 96 Spin Plate onto a new S-Block.

~60–80 min

Load ~650 µL into each well of the spin plate. Seal with sealing tape. Centrifuge at room temperature for 3 min at 4500 × g. Discard flow-through. Remove sealing tape.
Repeat loading and centrifugation until all supernatant has been processed. Discard final flow-through. Place spin plate back on the same S-Block.
Add 500 µL Solution EA to each well. Seal with sealing tape. Centrifuge at room temperature for 3 min at 4500 × g. Discard flow-through. Place spin plate back on S-Block.
Add 500 µL Solution C5 to the spin plate. Seal with sealing tape. Centrifuge for 3 min at 4500 × g.
Discard flow-through (remove any trace from S-Block). Place spin plate (still sealed) back on the same S-Block. Centrifuge again at room temperature for 5 min at 4500 × g. Discard flow-through.

~20 min

Carefully place the spin plate onto new collection microtubes. Discard sealing tape. Allow to air dry for 10 min at room temperature.
Add 100 µL Solution C6 to the center of each well. Seal with sealing tape. Centrifuge at room temperature for 3 min at 4500 × g. Discard sealing tape.
Seal the collection microtubes with caps provided. DNA is ready for downstream applications.

Storage

Store eluted DNA at −20°C for long-term use. Avoid repeated freeze-thaw cycles.

Stop and ask before:

Proceeding if the sealing film leaked during bead beating.
Using eluted DNA if A260/A280 or A260/A230 ratios are outside expected range (A260/A280 ~1.8, A260/A230 >1.5).
Skipping the 5 min air-dry step — residual ethanol from C5 will inhibit downstream reactions.