Instagene matrix genomic dna isolation kit

The phylogenetic status of the isolate was determined by genotyping the gene encoding 16S rRNA. In brief, the bacterial genomic DNA was isolated using the InstaGeneTM Matrix Genomic DNA isolation kit (cat # 732-6030 Bio-Rad Laboratories Pvt Ltd), and the 16S rRNA gene fragment was amplified by polymerase chain reaction (PCR) with a forward primer (Sense - 5′-AGAGTTTGATCMTGGCTCAG-3′) and a reverse primer (Antisense – 5′-TACGGYTACCTTGTTACGACTT-3′) using MJ Research Peltier Thermal Cycler. The PCR product was purified with Montage PCR Clean up kit (cat # P36322, Millipore) and the DNA sequence was determined using Illumina hiseq sequencer (YAAZH XENOMICS, India). The 16S rRNA sequence was analyzed by using NCBI BLAST similarity search tool and the multiple sequence alignment (Phylogeny analysis) was performed with the closely related sequence of blast results. The program MUSCLE 3.7 was used for multiple alignments of sequences and the resulting aligned sequences were cured using the program Gblocks 0.91b (to remove alignment noise) [15 (link)]. The phylogeny analysis was done with PhyML 3.0 aLRT and HKY85 was used as a substitution model [16 (link)].