# Changes in Soil Chemical Properties Due to Long-Term
## 2 Materials and Methods
### DNA Extraction, PCR Amplification, Cloning, and Sequencing of Functional Genes  
DNA was extracted using a PowerSoil(r) DNA isolation kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer's instructions using \(\sim\)0.30 g of fresh soil sample. The extracted DNA was stored at \(-\)20 \({}^{\circ}\)C until used in downstream analyses. The quantity and the quality of the extracted DNA were determined by Nano Drop 2000 (Thermo Scientific) and gel electrophoresis. PCR amplification of methane related genes was carried out with the same PCR conditions used in qPCR, except that a final elongation step was added at 75 \({}^{\circ}\)C for 5 min and a hold temperature at 4 \({}^{\circ}\)C (Table S1). PCR-amplified products using genomic DNA extracted from compost-treated paddy soils were then used for cloning. The PCR products were purified using Biomedic(r) Gel & PCR Purification Kit (Biomedic Co., Ltd., Bucheon, Korea). The purified PCR product was cloned into pTOP TA V2 vector using TOPclonerTM TA Kit (Enzymonics, Daejeon, Korea). The vector-insertligate was used to transform _Escherichia coli_ DH5\(\alpha\) competent cells (Biomedic Co., Ltd., Bucheon, Korea) and then _E. coli_ transformants were cultured on Luria-Bertani (LB) solid broth containing ampicillin/IPTG/X-gal for 12 h at 37 \({}^{\circ}\)C. White colonies were inoculated in LB liquid broth containing ampicillin (50 ug/uL) and then cultured in a shaking incubator (180 rpm) for 12 h at 37 \({}^{\circ}\)C. In order to identify recombinant clones, white colonies were subjected to colony PCR using vector primer sets, M13 forward, and M13 reverse primers. The PCR cycling condition was as follows: 5 min for initial denaturation at 94 \({}^{\circ}\)C, 25 cycles of 30 s at 94 \({}^{\circ}\)C, 30 s at 55 \({}^{\circ}\)C, 1 min at 72 \({}^{\circ}\)C, concluding with 1 cycle of 10 min at 72 \({}^{\circ}\)C. The PCR product was separated on 1.2% (_w/t_) agarose gel to check PCR amplification. Plasmid DNAs were isolated from the recombinant clones using Biomedic(r) Plasmid DNA Miniprep Kit (Biomedic Co., Ltd., Bucheon, Korea) following the manufacturer's protocol. Nucleotide sequences of the recombinant clones were determined using the two vector primers and dGTP BigDye(r) Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's guide. The obtained functional gene sequences were aligned and the affiliations were deduced using BLAST analysis using the NCBI ([[https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/)]([https://blast.ncbi.nlm.nih.gov/](https://blast.ncbi.nlm.nih.gov/))) [24]. Phylogenetic analyses were performed using MEGA version 6 [25] after multiple alignments of the data by CLUSTAL W [26]. DNA substitutions were done according to the Jukes and Cantor model [27] and clustering was performed using the neighbor-joining method [28]. The statistical confidence of the nodes was estimated by bootstrapping using 1000 replications [29]. Sequences of the _mcrA_ and _pmoA_ genes were deposited in GenBank and the accession numbers of the entries were OL739247 to OL739249 for _mcrA_ and OL691942 to OL691960 for the _pmoA_ gene sequences.