metagenome analysis of industrial effluents and identification of genes
Library construction using PCR Amplification was performed in two stages.The first stage involved the use of universal primers from the V3 region (341F CCTACGGGNGGCWGCAG) and V4 regions (805R GACTACHVGGGTATCTAATCC) of 16S ribosomal RNA, which were combined with indices. The components for the amplification included Hieff 2X PCR Master mix (15 l), In the first stage, a mixture consisting of Bar-Forward primer 1l, Reverse primer 1l, DNA approximately 10-20ng 3l, and distilled water 10lwas gently mixed, centrifuged, and placed in a 200 l PCR tube. The PCR conditions consisted of a preheat at 94C for 3 minutes, followed by 5 cycles of 94C for 30s, 45C for 20s, and 65C for 30s, and 20 cycles of 94C for 20s, 55C for 20s, and 72C for 30s. The reaction was completed with 72C for 5 minutes and 10C indefinitely. The second stage involved a similar process, but with different components. A mixture of Hieff 2X PCR Master mix, Forward primer, Index-PCR-Reverse primer, DNA, and distilled water was gently mixed, centrifuged, and placed in a 200 l PCR tube. The PCR conditions consisted of a preheat at 94C for 3 minutes, followed by 20 cycles of 94C for 20s, 55C for 20s, and 72C for 30s, and a final extension at 72C for 5 minutes and 10C indefinitely.The size of each resulting library was verified using 2% agarose gel electrophoresis. Overall, the PCR amplification process was carried out meticulously to ensure the production of high-quality libraries for downstream applications.