Omega e z n atm soil dna kit

DNA was extracted from 0.5 g of composite soil using the Omega E.Z.N.A^TM Soil DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, United States) according to the manufacturer’s instructions. The V4 region of the 16S rRNA gene was PCR amplified in triplicate using the following primers that targeted bacteria and archaea: 515F = (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R = (5′-GGACTACHVGGGTWTCTAAT-3′).
The primers were tailed with sequences to incorporate Illumina adapters with indexing barcodes. The PCR details and the related procedures were based on the 16S rRNA Amplification Protocol version 4-13 (Caporaso et al., 2012 (link)). The DNA concentrations were quantified, and the amplicons from each sample were pooled with an equimolar concentration for sequencing on an Illumina MiSeq Sequencer (San Diego, CA, United States) at the Chengdu Institute of Biology, Chinese Academy of Sciences, China.

DNA was extracted from wet soil (0.5 g) using the Omega E. Z. N. A TM Soil DNA Kit (Omega Bio-tek, USA), following the manufacturer’s instructions. Extraction was performed 3 times and the DNA extracts were pooled. The primer pairs ME1 (5″-GCMATGCARATHGGWATGTC-3″) and ME2 (5″-TCATKGCRTAGTTDGGRTAGT-3″) were used to specifically amplify a 760 bp-long mcrA (Hales et al., 1996 (link)), which was used to study methanogen communities. The 50 μl reaction mixture contained 1 μL of DNA template, 5 μL of buffer (10 ×), 2 μL of MgCl₂ (25 mM), 1 μL of deoxynucleoside triphosphates (10 mM), 1 μL of each primer (50 μM), and 2.5 U of Taq DNA polymerase (Takara). Polymerase chain reaction (PCR) conditions were as followers: initial denaturation at 5 min at 94^oC; followed by 30 cycles of 94^oC for 45 s, 50^oC for 45 s, and 72^oC for 2 min, and a final synthesis for 7 min at 72^oC (Bio-rad). The total PCR products were amplified three times and pooled. Amplicons were analyzed on 1% agarose gels with Goldview staining and purified using an Omega PCR purification kit (Omega Bio-tek, USA).