Gotaq green master mix

For validation of MiMIC conversion and CRIMIC cassette insertion events, the genomic DNA was extracted from ~20 adult flies using the PureLink Genomic DNA Mini Kit (Invitrogen). For MiMIC conversions, four reactions of PCR were performed with tag-specific primers and MiMIC specific primers as described previously (Diao et al., 2015 (link); Venken et al., 2011a (link)). The PCR reaction mix was: 1 μl genomic DNA (~10 ng), 1 μl primer 1 (10 μM), 1 μl primer 2 (10 μM), 4.5 μl H2O, and 7.5 μl GoTaq Green Master Mix (Promega). Hot start PCR conditions in C100 Touch Thermal Cycler (Bio-Rad) were: denaturation at 95° for 1 min, 34 cycles at 95° for 30 s, 56° for 30 s and 72° for 60 s, and post-amplification extension at 72° for 10 min. For CRIMIC cassette insertion, two reactions of PCR were performed with target-specific primers (see our website at Flypush) and attP-R primer (5’-CCCCAGTTGGGGC-3’) (Figure 2—figure supplement 1). PCR reaction mix was: 1 μl genomic DNA (~10 ng), 1 μl primer 1 (10 μM), 1 μl primer 2 (10 μM), 4.5 μl H₂O, and 7.5 μl GoTaq Green Master Mix (Promega). Hot start PCR conditions in C100 Touch Thermal Cycler (Bio-Rad) were: denaturation at 95° for 1 min, 40 cycles at 95° for 30 s, 56° for 30 s and 72° for 2 min 30 s, and post-amplification extension at 72° for 10 min.

Chlamydia trachomatis was amplified using a nested PCR protocol modified by Jalal et al. (2007) [33 (link)], which produced a sequence of 394 bps of the ompA gene of C. trachomatis. The first reaction used 6.0 μL of GoTaq Green Master mix (Promega, Madison, WI, USA), 0.5 μL (containing 20 pmol/μL of each primer) of the primers P1 (A) (5’GACTTTGTTTTCGACCGTGTT-3’) and P2 (5’AGCRTATTGGAAAGAAGCBCCTAA-3’), 2 μL of the genomic DNA, and 3 μL of sterile water for a final volume of 12 μL. The second reaction used 0.5 μL of the solution of the first reaction, 6.0 μL of Go Taq Green Master mix (Promega, Madison, WI, USA), 4.5 μL of sterile water, and 0.5 μL (20 pmol/μL) of the primers P3 (5’-AAACWGATGTGAATAAAGARTT-3̕) and P4 (5’-TCCCASARAGCTGCDCGAGC-3̕). In both steps of the nested PCR, negative and positive controls were used to optimize the results. Initial activation was conducted at 95°C in both stages of the PCR, but whereas this temperature was maintained for 5 minutes in the first stage, it was maintained for only 1 minute in the second stage. This activation was followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 54°C for 30 s, and extension at 72°C for 90 s, with a final extension step at 72°C for 7 min. The amplified products were visualized by electrophoresis in 1% agarose gel with 0.5 mg/mL of ethidium bromide.

To determine the strain types of all 49 E. coli isolates, genome fingerprints were obtained using enterobacterial repetitive intergenic consensus (ERIC) PCR [47 (link)] and CGG-PCR [48 (link)]. ERIC PCR amplification was performed using GoTaq^® Green Master Mix (Promega, Madison, WI, USA), ERIC1 and ERIC2 primers [47 (link)] at 0.5 µM each, 10 ng of DNA template, and cycling conditions of 95 °C for 7 min; 30 cycles of 94 °C for 1 min, 53 °C for 1 min, and 56 °C for 4 min; and a final extension at 65 °C for 16 min. CGG-PCR amplification was performed using GoTaq^® Green Master Mix (Promega, Madison, WI, USA), 0.5 µM N₆(CGG)₄ primer [48 (link)], 3.5 mM MgCl₂, 10 ng of DNA template, and cycling conditions of 95 °C for 3 min; 35 cycles of 95 °C for 1 min and 72 °C for 3 min; and a final extension at 72 °C for 8 min. The PCR products underwent electrophoresis, and DNA fingerprints were visualised on 2% agarose gels (1 × TBE buffer) with SYBR Safe DNA Stain (Invitrogen, Waltham, MA, USA).

UPEC virulence genes hlyA (alpha-hemolysin), fimH (type 1 fimbriae), papC (P-fimbriae), iutA (ferric aerobactin receptor), and cnf1 (cytotoxic necrotizing factor 1) were detected by PCR using primers listed in Table 1. The reaction was performed in a final volume of 25 µL, containing 12.5 µL Go Taq Green Master Mix (Promega), 100 ng of DNA, and 0.6 µM of each primer. The amplification conditions were: 1 cycle at 94 °C for 5 min, 25 cycles at 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 3 min, and 1 cycle at 72 °C for 10 min in a thermal cycler (Bio-Rad). The amplified products were analyzed by agarose gel electrophoresis (1.5%) stained with ethidium bromide and visualized in an ultraviolet transilluminator.
EAEC virulence genes aap (dispersin), aggR (transcriptional regulator), and the AA probe were detected by PCR using primers listed in Table 1. The reaction was performed in a final volume of 12.5 µL containing 100 ng DNA, 15 pmol of each oligonucleotide, and 5 µL of Go Taq Green Master Mix (Promega). The conditions used were 1 cycle at 94 °C for 5 min, 25 cycles at 94 °C for 30 s, 63 °C for 30 s and 72 °C for 3 min, and a final cycle at 72 °C for 10 min in a thermal cycler (Bio-Rad). The amplified products were analyzed by polyacrylamide gel electrophoresis (6%) stained with silver nitrate.