Amplicon synthesis, purification and sequencing

PCR amplification of the genomic DNA fragments covering questionable B. pumilus SAFR-032 genome regions was performed using Q5 High Fidelity PCR kit (New England Biolabs, Ipswich, MA). The reaction mixtures contained 0.16 μg of genomic DNA, 1 μM of each amplification primer and 40 μL of Q5 High Fidelity Master Mix in a final volume of 80 μL. After initial incubation at 94°C for 90 sec, the reactions were carried out for 37 cycles at 94°C for 30 sec, 52°C for 30 sec, and 72°C for 5 min. Final extension was performed at 72°C for 10 min. Then, the entire reaction mixtures were separated by electrophoresis on 1% agarose gel containing 0.2 μg/mL ethidium bromide. The bands of relevant size were excised from the gel, and the DNA was isolated from the gel slices on silica spin columns [31]. The purified rDNA amplicons were partially sequenced by the Sanger method at SeqWright, Inc (Houston, TX) using 5 primers for each amplicon: 16s_bpum_seq, 23s_bpum_seq1, 23s_bpum_seq2, and 2 amplification primers. Transposase gene amplicons were sequenced using amplification primers only.