Pcr clean up column kit

Bacterial genomic DNA was isolated from bacterial culture of selected bacterial isolate using Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacture instructions. By using 16 rRNA specific primers, forward (5`-AGAGTTTGATCCTGGCTCAG-3`) and reverse (5`-GGTTACCTTGTTACGACTT-3`), PCR reaction was performed as previously reported^{96 (link)}. Briefly, PCR reaction was started with initial denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1.5 min. An additional final extension step was carried out at 72 °C for 5 min. PCR amplified products were checked on 1.5% agarose gel electrophoresis, visualized under UV transilluminator, and purified by a PCR clean-up column kit (QIAGEN, Germany) for sequencing. Sanger sequencing of 16 rRNA gene was performed using a BigDye Terminator v3.1 Cycle Sequencing kit and a 3130xl Genetic Analyzer system (Applied Biosystems, USA). After the sequencing process, the annotated nucleotide sequence was analyzed using NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and deposited in Genbank. The phylogenetic tree was constructed based on the UPGMA statistical method with a bootstrap of 2.000 replicates using the MEGA 5 software^{97 (link)}.