Computational protocol: NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand

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[…] Genomic DNA was extracted from the cultured bacteria as described by Ausubel et al. (). Fragments of the nirK and nirS genes were amplified using the primer pairs nirK1F-nirK5R for nirK and nirS1F-nirS6R for nirS (Braker et al. ) in a final volume of 50 μl containing 5 μl of 10× PCR buffer 200 μM of deoxyribonucleoside triphosphate 1.0 U of Taq polymerase 1 μM of both primers, 400 ng of bovine serum albumin μl−1 and 10–100 ng of extracted gDNA. PCR conditions consisted of 1 min at 95°C, followed by a 1 min primer-annealing step and 1 min at 72°C. After 35 cycles, a final 7 min incubation at 72°C was performed. Expected amplicons were cloned using the PCR-Script™ Amp Cloning Kit (Stratagene, USA), and transformant selection was performed according to the manufacturer’s instructions. Selected transformants were then screened for the correct plasmid inserts by nested PCR, using two sets of primers (Braker et al. ). Plasmids were extracted from transformants that contained the desired inserts by the Fast Plasmid™ Mini kit (Eppendorf, USA). DNA sequencing was performed commercially by pyrosequencing at Macrogen, Korea.For each of the two gene fragments, the sequences were “blasted” and aligned by the National Center for Biotechnology Information BLASTN program and the ClustalX programmes, respectively. Phylogenetic analysis was performed with a neighbor-joining algorithm and distance calculation according to Jukes and Cantor using PAUP4.0*b. Neisseria gonorrhoeaeaniA gene (accession no. M97926) and the nirN gene from Pseudomonas aeruginosa (accession no. D84475) were used as out groups for nirK and nirS, respectively. The tree topology was evaluated by bootstrap analysis using 1,000 replicates. ClustalX programs, respectively. Phylogenetic analysis was performed with a neighbor-joining algorithm and distance calculation according to Jukes and Cantor using PAUP4.0*b. […]

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