Computational protocol: Occurrence, diversity and community structure of culturable atrazine degraders in industrial and agricultural soils exposed to the herbicide in Shandong Province, P.R. China

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Protocol publication

[…] The genes for 16S rRNA were amplified with the primer pair 63KWf/1387r. The PCRs were performed in a total volume of 50 μL containing 5 μL of TaKaRa 10× Ex Taq Buffer (Takara Biotechnology (Dalian) Co., Ltd., China), 2.0 mM MgCl2, 250 μM of each dNTP, 0.5 μM of each primer, 0.5 U of TaKaRa Ex Taq polymerase (Takara Biotechnology (Dalian) Co., Ltd., China) and 1.25 μL of a bacterial lysate as a template. The PCRs were started by denaturation at 95 °C for 3 min.; and consisted of 30 cycles: 94 °C for 1 min., 55 °C for 1 min., 72 °C for 2 min.; followed by extension at 72 °C for 5 min. The amplification products were analyzed by electrophoresis on an agarose gel in 0.5 × TBE. Target fragments of about 1.3 kb were cut out from the gel, purified and sequenced in the manner described for the fragments of atrazine degradation genes.The BLASTn similarity search was performed against 16S ribosomal RNA sequences database of GenBank. The phylogenetic analysis was performed using the MEGA5 software package []. 16S rRNA gene nucleotide sequences of known atrazine-degrading bacteria and species type strains sharing more than 98 % sequence similarity with the analyzed bacteria were included in the datasets. Multiple alignments were implemented using the CLUSTALW aligner of MEGA5 and then refined by hand. Phylogenies were inferred using the Neighbor-Joining algorithm with elimination of all positions containing gaps and missing data. […]

Pipeline specifications

Software tools BLASTN, MEGA, Clustal W
Application Phylogenetics
Organisms Zea mays, Phragmites australis
Chemicals Atrazine