4.2. DNA Extraction

A single nematode was placed into 10 µL of sterilized distilled water in a PCR tube and frozen in liquid nitrogen. Then, the nematode was crushed with a sterilized glass rod. PCR buffer (10 × 8 µL; Takara-Bio, Shiga, Japan) and 2 µL of Proteinase K solution (600 µg/mL, Roche, Basel, Switzerland) were added to the sample in the PCR tube, and then the mixture was frozen at −80 °C for 2 h. The PCR tube was then incubated at 65 °C for 1.5 h, followed by incubation at 95 °C for 10 min, and it was finally centrifuged at 10,000× g for 1 min. The supernatant DNA suspension was stored at −20 °C. Pure genomic DNA was also isolated from 10,000 H. schachtii J2s; nematodes were frozen in liquid nitrogen and ground with a mortar and pestle until they were completely homogenized. DNA was extracted from the resulting macerate using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). For DNA extraction from plant root samples, approximately 0.2 g of root was cut, frozen in liquid nitrogen, and ground to powder; then, genomic DNA was obtained by adding solvents from a Universal Genomic DNA Extraction Kit (Takara-Bio, Shiga, Japan), according to the manufacturer’s instructions. To isolate nematode DNA from artificially infested soil and natural field soil, soil samples were mixed, and a 10 g portion was added to a PowerBead tubes containing homogenizing and lysis buffer, before gently vortexing to mix. Then, total genomic DNA was extracted using the PowerMax Soil DNA Isolation Kit (No. 12988-10, Qiagen), according to the manufacturer’s protocol. DNA was quantified using the Nano Drop ND-2000 Spectrophotometer (Thermofisher, Waltham, MA, USA).