Soil

The DNA extraction procedure follows the lab SOP for the ‘Characterization of Prokaryotic and Eukaryotic Biodiversity from Soil Samples’ (Chaves et al., 2025a) within Biodiversity Genomics Europe project. This is mirror for the workflow hosted in WorkflowHub (which hosts the downloadable PDF).

Qiagen MagAttract® PowerSoil® Pro DNA Kit extraction method to isolate eDNA from soil samples using the KingFisher Apex instrument, following both the provider’s protocol and adjustments from the Earth Microbiome Project’s High Throughput DNA extraction protocol.

Equipment and Consumables

Items	Quantity	Storage
Soil samples	250 mg per sample	-20°C
Qiagen MagAttract PowerSoil Pro DNA Kit
Solution CD2		2-8º
Other reagents		15-25ºC
Proteinase K (PK)		-20 ºC
PowerBead Pro Plates or PowerBead Pro Tubes	1 for 96 samples or 1 per sample	15-25ºC
Consumables for KingFisher*: 96 deep-well plates 96-tip combs for deep-well magnets 96 microplates (200 µL)	6 for 96 samples 1 for 96 samples 1 for 96 samples
Laminar flow hood
Vortex + adaptors
Reservoirs with lid	4
80% Ethanol	1500µL per sample
100% ethanol	for Buffer QSB1
100% Isopropanol	for Buffer MW1
Low-bind microplate	1 for 96 samples

* Thermo Scientific KingFisher Apex Purification System

Working space

• Dedicated DNA extraction room or laminar flow hood.

• Clean the working space and material with disinfectant and ethanol and leave the UV-light on for at least 15 mins.

• For each 96 extraction plate leave at least one well for a PCR negative control (PNC) per plate. And add one extraction negative control (ENC) per batch (e.g 46 samples + 1 ENC)

• Use extra-long pipette tips (1000–1250 µL);

• Use filtered tips at all steps.

DNA extraction

Preparation

• Turn on the oven at 65ºC or directly to 56ºC if not proceeding with step 2;

• For each 96-well plate to be processed:

  • add 400 µL RNase A Solution to 80 mL Solution CD1;

1. Spin the PowerBead Pro Tubes with the sample and add 800 µL Solution CD1/RNase A to each tube.

   Note: if 250 mg of dry soil sample absorbs all the liquid from the tube (no supernatant left), then lower the amount of soil in this sample.

2. Incubate samples at 65ºC for 10 mins. Note: This step is optional but can improve lysis efficiency

3. Homogenize samples thoroughly using a vortex with a horizontal tube adapter for 25 min at maximum speed.

4. Spin the tubes, add 25 µL of PK (20 mg/mL) to each tube and vortex for 30s.

5. Spin the PowerBead Pro Tubes, and incubate at 56ºC for at least 3h (can be incubated overnight).

After incubation

• prepare Buffer QSB1 and Buffer MW1 according to the instructions on the bottles;

• Prepare fresh 80% ethanol;

• Prepare a reservoir with CD2 Solution, 30mL per full plate of extractions.

6. Centrifuge the PowerBead Pro Tubes at 15000 x g for 1 min.

7. Transfer the supernatant to the CMTRs and add 300 µL Solution CD2 to each well of CMTRs. Pipette up and down 3-5 times to thoroughly mix after supernatant addition to each well.

Note: Expect 500-600 µL. The supernatant may still contain some soil particles.

8. Seal the CMTRs with the caps provided and then vortex. Centrifuge the CMTRs at 4500 x g for 6 min at room temperature.

9. Taking care to avoid any residual pellet, transfer the supernatant from each well to a clean KingFisher deep-well 96 plate. Expect to recover 650-700µL maximum.

10. Resuspend MagAttract Suspension G Beads by vortexing. For each 96-well plate to be processed, add 3 mL of the resuspended MagAttract Suspension G Beads to 44 mL Buffer QSB1 and mix well. Immediately transfer to a multichannel pipette reservoir.

Note: Maintain the MagAttract Suspension G Beads in suspension (by pipetting up and down several times) to ensure uniform distribution.

11. Add 470 µL of the MagAttract Suspension G beads/Buffer QSB1 mix to each well-containing lysate in a KingFisher 96 deep-well plate and mix by pipetting (KF Plate 1).

12. Transfer up to 650 µL of the MagAttract Suspension G beads/Buffer QSB1/lysate mix from each sample to another KingFisher 96 deep-well plate (KF Plate 2), to be used in the first binding step. Both KF Plates 1&2 will be placed in the machine to improve yield.

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KingFisher Apex Purification System

1. Add 500 µL Buffer MW1 (MW1 Plate) to each well of one clean KingFisher 96 deep-well plate.

2. Add 500 µL fresh 80% ethanol to each well of three clean KingFisher 96 deep-well plates (EtOH Plates 1-2).

3. Add 100 µL Solution C6 (C6 Plate) to each well of a clean KingFisher 96 microplate.

4. Turn on the KingFisher Apex Purification System and load the robotic decks in the following order: Tip Comb, C6 Plate, EtOH Plates 1-3, MW1 Plate, KF Plate 2, and KF Plate 1 as indicated in the program.

5. Initiate the robotic program.

6. Upon completion of the robotic program, transfer the eluted DNA from the KingFisher 96 microplate to a low-bind microplate.

7. Assess the quantity and quality of DNA using fluorometry (Qubit) and spectrophotometry (Epoch) methods. Dilute all samples to 10 ng/µL.

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References

Chaves, C., Najera Cortazar, L. A., Martins, F., Anslan, S., Beja-Pereira, A., Magalhães, M., & Price, B. (2025a). Characterization of Prokaryotic and Eukaryotic Biodiversity from Soil Samples. WorkflowHub. https://doi.org/10.48546/workflowhub.sop.12.2

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