Genomic dna extraction kit for bacteria

The selected P. polymyxa strain APEC128 was subjected to molecular identification using sequence homology of its 16S rRNA gene (Weisburg et al., 1991 (link)). Genomic DNA of APEC128 was isolated using a Genomic DNA Extraction Kit for bacteria (iNtRON Biotechnology, Seongnam, Korea) following manufacturer’s instructions. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) with Taq DNA polymerase and primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). The conditions for thermal cycling were as follows: denaturation at 94°C for 5 minutes followed by 30 cycles at 94°C for 1 minute, annealing at 56°C for 1 minute and extension at 72°C for 1 minute. At the end of the cycling, the reaction mixture was held at 72°C for 5 minutes and then cooled to 4°C. The obtained PCR products were sequenced with an automated sequencer (Genetic Analyzer 3130; Applied Biosystems, Foster City, CA, USA) using the same primers. The sequences were compared for similarity with the reference species of bacteria available in genomic database using the National Center for Biotechnology Information (NCBI)-BLAST tool. Sequence alignment and phylogenic tree construction were performed using a MEGA 4.0 program (Tamura et al., 2007 (link)).