How to effectively extract soil DNA | Dealing with challenging microbiome samples

Briefly spin the power bead tubes to settle beads, then transfer soil using weighing paper or funnel into the tube without overfilling. Optionally store tubes overnight in freezer and allow to reach room temperature before proceeding.

Add solution CD1 to the power bead tubes to disperse soil, dissolve humic acids, and protect DNA from degradation. Briefly vortex the sample to mix thoroughly.

Place tubes onto balanced tube adapters and load into the tissue lyzer instrument. Run disruption at 25 Hz for 5 minutes using the soil DNA protocol, then reorient tubes and repeat with the same parameters.

Centrifuge tubes to pellet solids and transfer supernatant to new 2 mL microcentrifuge tubes. Add solution CD2 to precipitate decontaminants that interfere with downstream applications.

Add solution CD3, vortex for 5 seconds, and load the lysate mixture onto an MB spin column. Centrifuge and pass through twice, then add solution EA to remove proteins and other non-aqueous contaminants.

Add solution C5 to further purify DNA and repeat the washing step. Add another round of C5 to ensure thorough removal of ethanol, which interferes with downstream applications.

Place the MB spin column onto an elution tube and add solution C6 at the center of the membrane to ensure complete release of DNA. Centrifuge to collect the purified DNA.

Freeze the extracted DNA for storage, as solution C6 does not contain EDTA. The DNA is now ready for downstream applications or can be referenced with troubleshooting guides if needed.