Gene ruler 1 kb dna molecular weight marker

PCR assays were performed to determine the capsular serotypes for K1 or K2 and another 11 virulence encoding genes, including iucA, entB, uge, magA, terW, iroB, kfuB, rmpA, rmpA2, wabG, and irp2. PCR reaction mixtures were prepared in total volumes of 20 μL. Each reaction contained 1 μL of template DNA, 1 μL (equivalent to 10 pmol concentration) of each primer, and 10 μL of GoTaq^® Green Master 2 × Ready Mix (Promega, Madison, WI, USA). The volume was completed to 20 μL by 7 μL of nuclease-free water. The PCR amplification conditions were as follows: initial denaturation for 5 min at 95 °C, then 35 cycles of denaturing at 95 °C for 30 s, annealing for 30 s and extension at 72 °C, followed by a final extension at 72 °C for 7 min. The appropriate annealing temperature for each pair of primers and the time for the extension step for each PCR amplicon are mentioned in Table S1. DNA fragments of PCR products were detected through TAE agarose gel (1 %) (Bioline, London, UK) electrophoresis in 1 × TAE buffer (Thermo Scientific, Waltham, MA, USA). The Gene Ruler 1 kb DNA molecular weight marker (Thermo Scientific, Waltham, MA, USA) was used for sizing the PCR products.