.. |logo_BGE_alpha| image:: _static/logo_BGE_alpha.png :width: 300 :alt: Alternative text :target: https://biodiversitygenomics.eu/ .. |eufund| image:: _static/eu_co-funded.png :width: 200 :alt: Alternative text .. |chfund| image:: _static/ch-logo-200x50.png :width: 210 :alt: Alternative text .. |ukrifund| image:: _static/ukri-logo-200x59.png :width: 150 :alt: Alternative text .. |logo_BGE_small| image:: _static/logo_BGE_alpha.png :width: 120 :alt: Alternative text :target: https://biodiversitygenomics.eu/ .. raw:: html .. role:: red |logo_BGE_alpha| .. _DNAex_soil: 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 || 1 for 96 samples || 15-25ºC | || Pro Tubes || or 1 per sample || | +-----------------------------------------+-------------------+----------+ || Consumables for KingFisher*: || || | || 96 deep-well plates || 6 for 96 samples || | || 96-tip combs for deep-well magnets || 1 for 96 samples || | || 96 microplates (200 µL) || 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 -------------- .. admonition:: 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). .. admonition:: 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. ___________________________________________________ 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**. ____________________________________________________ **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 ____________________________________________________ |logo_BGE_small| |eufund| |chfund| |ukrifund|