16S rRNA gene sequencing of hydrocarbon-degrading bacteria: isolates OD7, OD11, and OD15 from used engine oil-contaminated soil in Kozhikode, India
Genomic DNA was extracted from purified bacterial cultures using the NucleoSpin Tissue Kit (Macherey-Nagel) according to the manufacturers instructions. The extracted DNA was assessed for quality and integrity via 0.8% agarose gel electrophoresis. The 16S rRNA gene was amplified using gene-specific primers 16S-RS-F (5-CAGGCCTAACACATGCAAGTC-3) and 16S-RS-R (5-GGGCGGWGTGTACAAGGC-3), targeting conserved regions of the 16S rRNA gene. PCR amplification was carried out using a GeneAmp PCR System 9700 (Applied Biosystems) and 2X Phire Master Mix under the following conditions: initial denaturation at 95C for 5 min, followed by 35 cycles of 95C for 30 s, 60C for 40 s, and 72C for 60 s, with a final extension at 72C for 7 min. PCR product cleanup was performed using ExoSAP-IT (USB), and sequencing was conducted using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), followed by ethanol-sodium acetate precipitation. The cleaned sequencing reaction was analyzed on an ABI 3500 Genetic Analyzer using Sanger dideoxy sequencing technology. Resulting chromatograms were evaluated using Sequence Scanner Software v1 (Applied Biosystems), and sequence editing and alignment were carried out using Geneious Pro v5.1.