Mo bio s powersoil dna isolation kit

The DNA extraction were performed as described by Song et al., 2020 (link). DNA (0.5 g) was extracted from rhizosphere soil and root samples using the MO BIO’s PowerSoil DNA isolation kit (Qiagen, Germany) according to the manufacturer’s protocol. The maize roots were ground in liquid nitrogen prior to DNA extraction. A NanoDrop 2000 spectrophotometer (Waltham, Massachusetts, United States) was used to assess the concentration of the extracted DNA. The bacterial and fungal communities were investigated by targeted amplification of the V4 region of the bacterial 16S rRNA and the fungal ITS region, respectively. The selected regions were amplified using universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT; for bacterial 16S rRNA) and 5.8SFun (AACTTTYRRCAAYGGATCWCT) and ITS4Fun (AGCCTCCGCTTATTGATATGCTTAART; for fungal ITS region). The PCR mix included 1 μl DNA, 2.5 μl forward and reverse primers, and 5 μl PCR buffer. PCR was performed as follows: denaturation for 3 min at 95°C; followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 45 s; and a final extension at 72°C for 10 min. The PCR reaction procedures and product purification were performed as described by Taş et al. (2014) (link). The amplicon libraries were sequenced using the Illumina MiSeq platform (Illumina, USA) with a paired-end sequencing strategy.

Total rhizosphere genomic DNA was extracted from 0.3 g of well-mixed rhizospheric soil (wet weight) using a MO BIO’s PowerSoil DNA Isolation Kit (QIAGEN, Toronto, ON, Canada), following the manufacturer’s instructions. The roots were cleaned with tap water to remove soil particles and then crushed in liquid nitrogen using a pestle and mortar. The roots’ total DNA (including plant and endophytic DNA) was extracted from 0.1 g of well-mixed wet material using a DNeasy Plant Mini Kit (QIAGEN, Toronto, ON, Canada), following the manufacturer’s instructions. DNA concentrations were measured using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Scientific, Wilmington, DE, USA). All extracts were stored at −20 °C before PCR processing.