Mupid exu system

Bacterial genomic DNA samples were extracted using ZymoBIOMICS DNA Kits (Zymo Research, CA, USA), following the manufacturer’s instruction. The PCR reaction mixture (25 µL) contained a 1× JumpStart REDTaq ReadyMix (Sigma) and each primer (Table 2). The list of primers used in the mPCR are also presented in Table 2 [34 (link),35 (link),36 (link),37 (link)]. The PCR reaction was performed as follows: initial activation of DNA polymerase at 95 °C for 3 min, plus 35 cycles of denaturation at 95 °C for 30 sec, primer annealing and extension at 62.5 °C for 1.30 min, and a final extension at 72 °C for 5 min. A negative control was included in each run, consisting of the same reaction mixture but with water instead of template DNA.
The PCR products (5 µL) were analyzed using gel electrophoresis on 1.5% (w/v) agarose gel in 0.5 × TBE buffer at a constant voltage of 100 V for 30 min (Mupid exU system; Takara; Tokyo, Japan). The gels were stained with ethidium bromide and visualized under ultraviolet light (GeneGenius Bioimaging System; SynGene; Cambridge, UK). The sizes of the PCR products were determined by comparison with molecular-sized standards (GeneRuler™ 100 bp Plus DNA ladder; Thermo Fisher Scientific; Vilnius, Lithuania).