100 bp dna ladder

Several genes are known to be associated specifically with APEC. In this study, we selected the fimC, iucD, and papC genes as the molecular markers for the detection of APEC. These are the commonly APEC-associated virulence genes detected in the majority of the studies focused on APEC (8, 22, 60, 61, 62, 74). Moreover, as mentioned earlier, iucD differentiates APEC from non-APEC (17). Once confirmed, isolated E. coli were screened by PCR for detecting the APEC-associated virulence genes fimC, iucD, and papC [12 (link)]. The primers used for the detection of APEC are listed in Table 1.
PCR tests were done in a final 25 µL reaction with 12.5 µL of master mix (2X) (Promega, Madison, WI, USA), 4 µL of genomic DNA (50 ng/µL), 1 µL of each primer, and 6.5 µL of nuclease-free water. After completion, the amplified PCR products were analyzed by electrophoresis in 1.5% agarose. Amplicons were stained by ethidium bromide and visualized under an ultraviolet trans-illuminator (Biometra, Göttingen, Germany). A 100 bp DNA ladder (Promega, Madison, WI, USA) was used to check the size of the PCR amplicons.

For each sample, PCR amplification was performed in a reaction volume of 25 μL containing 20–50 ng of DNA, 10X DreamTaq Green Buffer (Thermo Fisher Scientific, Waltham, MA, United States) with final concentrations of 4.5 mM MgCl₂, 0.2 mM of dNTPs, and 0.75 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific). Specific published primers, designed for related Prunus species, were applied for the amplification of target sequences (Table 1). The PCR products were separated on 1% TBE agarose gels at 80 V for 30 min and DNA bands were stained with ethidium bromide. Fragment sizes were estimated by comparison with the 100-bp DNA ladder (Promega, Mannheim, Germany).
PCR products were cloned into the pTZ57R/T plasmid vector using the InsTAclone PCR Cloning Kit (Thermo Fisher Scientific) and JM109 competent cells, isolated with GeneJET^TM Plasmid Miniprep Kit (Thermo Fisher Scientific) and sequenced using an ABI 3500 XL Genetic Analyzer (Applied Biosystems, Foster City, CA, United States).

For PCR amplification, a total of 50 µl reaction mixture contained 28 µl Go taq green master mix (Promega, Germany), 20 µl RNase free water, and 1 µl of each forward and reverse primer was prepared. The primer used for coa gene amplification as described by Hookey et al. [13 (link)] was 5’ATA GAG ATG CTG GTA CAG G3’and 5’GCT TCC GAT TGT TCG ATG C3’. A total of 2.5µl of DNA template were added to the mixture. The mixture then amplified using PCR cycler according to the protocol of Akineden et al. [14 (link)] with modification as following: Predenaturation at 94°C for 45 s, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min. The amplification ended by a final extension at 72°C for 2 min. The presence of PCR products was determined by electrophoresis of 10 µl of products in 2% agarose gel with TBE buffer and 100 bp DNA ladder as a marker (Promega, Germany).