DNA extraction, PCR amplification for bacterial isolates
The DNA extraction from pure colonies of bacterial isolates was carried
by using GenElute™ Bacterial Genomic DNA Kit Protocol, Sigma, following
the manufacturer’s protocol. Eluted DNA samples were checked in agarose
gel. 16S rRNA gene was amplified in an Eppendorf Mastercycler using
primer pair 27f (5’ -AGAGTTTGATYMTGGCTCAG) (24) and 1401r (5’ -
CGGTGTGTACAAGACCC) (25). In brief, PCR amplification was performed in a
25 µl reaction mixture containing 1x PCR buffer, 1.5 mM
MgCl2, 200 µM dNTPs, 10 pmol of each primer, 1 U of Taq
DNA polymerase, and 30–50 ng of gDNA. The cycling conditions were as
follows: initial denaturation at 95 °C for 5 min. This was followed by
35 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 60 s, and a final
extension at 72 °C for 10 min. The amplicons were resolved on 1.5 %
(w/v) agarose gel containing 0.05 % ethidium bromide along with a 100
bp ladder for size and mass calculation of PCR products and visualized
under Chemi-Doc System (BioRad). The amplified PCR products were
purified using the Gen-elute PCR cleanup kit (Sigma). The purified PCR
products of bacterial gene fragments were sequenced at the
1st Base sequencing company, Singapore using an
automated DNA sequencer. Contigs were assembled based on their phred
scores (>15) and identified by aligning in the NCBI
reference rRNA database using the blastn algorithm.