Taq polymerase kit

Initially, amplifications were performed using primers OPB-15, OPB-17 and P1254 (S1 Table), previously described by Lin et al. (1996) [12 (link)]. Due to the lack of variability shown by the OPB-17 and P1254 primers (data not shown), only OPB-15 was used to analyze the complete Salmonella collection. The amplification reaction was performed in 40 μl containing 1.5 U of Taq polymerase, 3 mM MgCl, 0.2 mM dNTPs, 2 μM of primer and, as template, 2 μL (roughly 50 ng) of extracted total DNA of each strain, using a commercial Taq polymerase kit (Thermo scientific). The cycling program was as follows: 3 min at 95°C, 3 min at 35°C and 3 min 30 s at 72°C for 3 cycles, followed by 25 cycles of 30 s at 94°C, 1 min at 35°C and 1 min 30 s at 72°C, and completed by a final extension for 5 minutes at 72°C. The amplification products were resolved in 1% agarose gels in 0.5X TAE buffer. Fragment size was determined by comparing with a 100 bp ladder (Thermo).

Based on available pEAF sequences, primers specific for the amplification of the repFIIA replication region and of two distant regions within the tra operon (traI and traC genes, encoding for the relaxase, and an ATPase required for pilus production of the type IV secretion system, respectively), were designed using the primers4clades web server [34 (link)] (Table 2). For the detection of the bfp operon, the bfpA sequence was amplified using the primers described by Lacher et al. [35 (link)]. DNA was extracted from liquid cultures by a modification of the salt extraction method described by Miller et al. (1988) [36 (link)]. Amplifications were performed in 50 μl reactions using a commercial Taq polymerase kit (Thermo scientific) and 1.5 U Taq polymerase per tube, with a final concentration of 1.5 mM MgCl, 0.2 mM dNTPs, and 0.5 μM each primer. Two μL of extracted total DNA was used as a template (roughly 50 ng). The cycling program was as follows: 5 min 95°C followed by 30 cycles of 45 s at 94°C, 30 s at 57°C, and 45 s at 72°C and completed by a final extension for 5 minutes at 72°C.

To express the insecticidal genes, phloem-limited promoters were cloned from a Pakistani BBTV isolate40 (link). Two viral components, the capsid protein (CP) and the nuclear shuttle protein (NSP) were chosen for amplifying the promoters. The intergenic region (IR) was considered as the promoter and sequence-specific primers (Table 1 in supplementary data) were used to amplify the whole IR by PCR. The deleted part of the NSP intergenic region was used as reported45 (link). The PCR reaction mixture was prepared using the Thermo Scientific, USA Taq polymerase kit, containing 1X reaction buffer, MgCl₂, 2 mM dNTPs, 10 pmol of each primer, 10 ng of template and 0.5 units of Taq polymerase. The PCR reaction was run with the following temperature profile: initial denaturation temperature 94 °C for 5 min, 1 cycle; followed by 35 cycles of 94 °C for 30 sec, 50 °C for 30 sec, 72 °C for 45 sec; and final extension at 72 °C for 10 min. SacI and HindIII restriction sites were used for cloning into the pJIT166 expression vector67 (link), replacing the 2X 35S promoter upstream of the GUS reporter gene. In the next step, the complete expression cassette from pJIT166 was excised and cloned into the pGreen0029 binary vector68 (link) at restriction sites SacI and XhoI, which makes the construct ready for further cloning of genes under this promoter.