Engine t100 thermal cycler

The bacterial genomic DNA was extracted using a genomic DNA extraction kit (Invitrogen, USA) from pure cultures. A NanoDrop spectrophotometer was used to measure the DNA concentrations. For the invA gene, PCR was carried out using the forward (GTG AAA TTA TCG CCA CGT TCG GGC AA) and reverse (TCA TCG CAC CGT CAA AGG AAC C) oligonucleotide primers with a reaction volume of 25 μL, containing: 8.5 μL nuclease-free water, 12.5 μL PCR Master Mix, 2 μL template DNA, and 1 μL of each primer utilizing an Engine T100 ThermalTM cycler (BioRad, Singapore). The thermal cycling conditions included an initial step of denaturation at 94°C for 5 minutes, then 30 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 70 minutes, followed by a single, concluding extension step at 72°C for 7 minutes [13 (link)].

Polymerase chain reactions were conducted using the Universal primers: forward (5’-AGA GTT TGA TCC TGG CTC AG-3’) and the Reverse (5’-ACG GCT ACC TTG TTA CGA CTT-3’), with the reaction volume of 25 µL containing 12.5 µL PCR Master Mix, 2 µL template DNA, 8.5 µL nuclease free water and 1 µL of each oligonucleotide primer using an Engine T100 Thermal^TM cycler (Bio-Rad, Singapore). The thermo cycling conditions consisted of an initial denaturation step at 95 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s and extension at 72 °C for 5 min, and finally a single and final extension step at 72 °C for 7 min. Polymerase chain reaction products were identified by electrophoresis on 2% (weight per volumne [w/v]) agarose gel stained with ethidium and visualised under ultraviolet light on a ChemiDoc Imaging System (Bio-Rad ChemiDocTM MP Imaging System, Hertfordshire, United Kingdom).