Dna wizard genomic dna purification kit

Identification of 16S rRNA gene was initiated by culturing isolates of Bacillus sp. ES4.3 into 20 mL of Luria Bertani medium, incubated at 35 o C with agitation at 120 rpm for 48 hours. Furthermore, to obtain DNA, extraction was carried out using the CTAB method (Ausubel et al. 2003) with DNA Wizard Genomic DNA Purification Kit (Promega). DNA purity and concentration values were measured using Multiskan GO on λ 260 nm and λ 280 nm. Hereafter, 16S rRNA gene amplification was carried out using Eppendorf Mastercycler equipment. This process begins by adding GoTaq Green Master Mix and 16S rRNA primers, in primers 27F and 1492R. The Polymerase Chain Reaction (PCR) was conditioned as follows: initial denaturation of 94 o C for 2 minutes, denaturation of 92 o C for 30 seconds, annealing 55 o C for 30 seconds, elongation of 72 o C for 1 minute, final elongation of 72 o C for 5 minutes, 35 cycles. The PCR product was visualized through an electrophoresis process using 1% agarose gel followed by Ethidium Bromide staining and observed in ultraviolet light. The PCR samples were sent to the 1 st Base DNA Sequencing Service Malaysia. Amplicon was sequenced and analyzed for similarity with GenBank data using BLASTn NCBI (Altschul et al. 1997) (link). The data were also analyzed for their relation by building a phylogenetic tree using MEGA version 6 software (Tamura et al. 2013) (link).