Soil DNA Extraction Modified Protocol for Dryland Agroecosystems

Soil samples should be stored cold, at least -20 C (ideally at -80 C).

Fill a 12 ml homogenization vial approximately 2/3 full with soil (Spex SamplePrep 6133PC-T).  Include three 6.35 mm diameter chrome steel beads (BioSpec Products Cat. No. 11079635c).

Homogenize at 4000 rpm for 0h 0m 10s using the SPEX SamplePrep 1200 Genolyte homogenizer with the single-vial attachment for 12mL vials.  Re-homogenize as needed at 4000 rpm for 0h 0m 10s to avoid the soil from thawing and sticking together.

This protocol is modified from the Qiagen manufacturer protocol for the DNeasy PowerLyzer PowerSoil Kit.

Equipment

Value	Label
DNeasy PowerLyzer PowerSoil Kit	NAME
DNA Extraction Kit	TYPE
Qiagen	BRAND
12855-50	SKU

.

Add approximately 80µL of soil sample by volume to the PowerBead Tube provided.

Add 750µL of PowerBead Solution to the PowerBead Tube.

Add 60µL of Solution C1 and invert several times or vortex briefly.

Bead beating: Spex 1200 Genolyte homogenizer: Place the PowerLyzer Glass Bead Tubes into the tube holder attachment for the homogenizer (holds 6). The PowerBead Tubes must be balanced on the tube holder. Run the samples at a time and RPM suitable for your soil type. For our samples, 3000 rpm for 0h 0m 35s .

Centrifuge Bead Tubes at 10,000 x rcf for 0h 3m 0s. Note: We increased the centrifugation time to ensure that no sediment/soil is left in suspension, improving DNA quality downstream.

Transfer the supernatant to a clean 2 ml collection tube. Note: Expect 400-500µL. Supernatant may still contain some soil particles.

Add 250µL of Solution C2 and vortex for 5 sec. Incubate at 2-8°C for 5 min.

Centrifuge the tubes at 10,000 x rcf for 1 min. Avoiding the pellet, transfer up to 600µL of supernatant to a clean 2 ml collection tube.

Add 200µL of Solution C3 and vortex briefly. Incubate at 2-8°C for 0h 5m 0s .

Centrifuge the tubes at 10,000 x rcf for 0h 3m 0s. Avoiding the pellet, transfer up to 750µL of supernatant to a clean 2 ml collection tube. Note: We increased the centrifugation time to ensure that no sediment/soil is left in suspension, improving DNA quality downstream.

Add 1200µL of Solution C4 to the supernatant and vortex for 0h 0m 5s.

Load 675µL of the supernatant onto a MB Spin Column and centrifuge at 10,000 x rcf for 0h 1m 0s. Discard the flow through and add an additional 675mL of supernatant.

Centrifuge at 10,000 x rcf for 0h 1m 0s. Load the remaining supernatant onto the MB Spin Column and centrifuge at 10,000 rcf for 0h 1m 0s. Note: A total of three loads for each sample processed are required.

Add 500 μl of Solution C5 and centrifuge at 10,000 x rcf for 0h 0m 30s. Discard the flow through. Repeat this step once more: add 500µL of Solution C5 and centrifuge at 10,000 x rcf for 0h 0m 30s. Discard the flow through. Note: We repeated this washing step to minimize salt contamination, which helps improve DNA A260/230 values.

Centrifuge again at 10,000 x rcf for 0h 1m 0s.

Carefully place the spin filter in a clean 2 ml collection tube. Avoid splashing any Solution C5 onto the MB Spin Column.

Add 50µL of Solution C6 to the center of the white filter membrane. Note: We used the minimum recommended amount of Solution C6 to yield greatest DNA concentrations.

Centrifuge at 10,000 x rcf for 0h 0m 30s. Discard the MB Spin Column.

The DNA is now ready for downstream applications.