The broth cultures were grown at their respective temperature of the isolates with shaking at 200 rpm till the cultures reached OD600 of 0.4-0.5. Thereafter, cells were pelleted by centrifugation at 9167 × g for 10 min at 4°C and washed with TE buffer [10 mM Tris–HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)] and pellets were either frozen (-20°C) for storage or used immediately for genomic DNA extraction by using the method of Sambrook & Russell . DNA samples were quantified by running on agarose gel electrophoresis
using 0.8% agarose gel in 1 × tris-boric acid EDTA (TBE) (89 mM tris pH 7.6, 89 mM boric acid, 2 mM EDTA) and visualized by ethidium bromide selleck chemical (0.5 μg ml-1) staining, to determine DNA size and to assess RNA contamination. PCR Amplification and sequencing Amplifications were performed in 50 μl reaction mixture containing 75 ng of template DNA, 1-unit of i-Taq™ polymerase (NEB, UK), 2 mM MgCl2 (NEB, UK) , 2 μl of 10X PCR buffer, 0.1 mM dNTP (NEB, UK), 100 ng of each forward (8f’:5’-AGAGTTTGATCCTGGCTCAG-3’ ), and reverse (1542r’:5′-AAGGAGGTGATCCAGCCGCA-3’
) primers. The amplification was carried out using G-strom thermal cycler (Labtech, UK). Amplification programme consisted of initial cycle of denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, initial extension at 72°C for 1 min 30
sec and final extension at 72°C for 7 min. Amplified products were electrophoresed LY2109761 at 5 Vcm-1 through 1.5% agarose gel containing 0.5 μg ml-1 ethidium bromide in 1xTBE electrophoresis buffer with 50 bp DNA Ladder (NEB, UK). The gels were visualized under UV illumination in Gel Documentation system 2000 (Biorad, Hercules CA, USA) and stored as TIFF file format. Sizes of the amplicons were estimated in comparison with 50bp DNA ladder (NEB, UK). Sequencing of 16S rRNA gene and phylogenetic LY3023414 datasheet analysis The expected DNA band of 1.5 kb was excised from gel and purified very using the gel elution kit (Sigma-Aldrich, USA) as per the manufacturer’s protocol. Sequencing reactions were carried out with a BigDye Terminator cycle sequencing kit (Applied Biosystems, USA), standard universal primer forward (8f’) and reverse (1542r’) primer and sequenced by using ABI Prism 3100 genetic analyzer (Applied Biosystems, USA). The sequences thus obtained were assembled and edited using Clone Manager Version 5 (http://www.scied.com/pr_cmbas.htm). Database search was carried out for similar nucleotide sequences with the BLAST search of Non-reductant (NR) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) ). Partial length 16S rRNA gene sequences of strains closely related to the isolate were retrieved from NCBI for further analysis.