Mytaq hs mix

Bacterial DNA was extracted by thermal lysis. A 1 µl loop of culture material was suspended in 45 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA) and 5 µl lysostaphin (0.1 mg/µl). After incubation for 45 min at 37 °C, the sample was heated at 95 °C for 10 min and cooled by adding 200 µl TE buffer.
For the detection of virulence genes, the following primers were used: (sak-F, 5′-GTAAGTGCATCAAGTTCATTCG-3′; sak-R, 5′-GCTCTGATAAATCTGGGACAAC-3′; scn-F, 5′-AATGGCTCTTCTTCGCTTTC-3′; scn-R, 5′-TGCTACTTTATTCCTTACGGC-3′; chp-F, 5′-GTTTTTTAACGGCAGGAATCAG-3′; chp-R, 5′-TTTCTATCTTCAGCAAGTGGTG-3′), (hlb1B, 5′-GTTGCAACACTTGCATTAGCA-3′; hlb2, 5′-TGTGTACCGATAACGTGAAC-3′); (pvl-F, 5′-TTGAAATGTTGTACTTAGAACC-3′; pvl-R, 5′-TAGGTAAAATGTCTGGACATG-3′). Primers used for the detection of the enterotoxin A gene sea and phage integrase genes have previously been described by Johnson et al.^{41 (link)}, Goerke et al.^{11 (link)} and Kahankova et al.^{42 (link)}. PCR was performed using 2x MyTaq HS Mix (Bioline, Luckenwalde, Germany) with a final volume of 25 µl containing 10 µl MyTaq HS Mix, 1 µM of forward and reverse primer, 1 µl DNA (10 ng) and dH₂O.
For the determination of the attB site of ΦSa3 phage P282 within hlb, PCR products of the β-hemolysin gene were purified using the Invisorb Spin DNA Extraction Kit (Stratec Molecular, Berlin, Germany) and sequenced (Eurofins Genomics, Ebersberg, Germany).

Core genes of the two most prevalent groups of strains observed in this present study were used to design pairs of discriminating primers, with conserved regions within core genes used to identify sites for the anchoring primers. Then, whole genomes were scanned to confirm the specificity of the primer sets using ecoPrimer v0.5 (Riaz et al., 2011 (link)). Candidate PCR primer pairs (targeting sequences in yjcS and intA_5 genes) were evaluated against a panel of field isolates and reference and type strains to confirm their specificity (Supplementary Table 2). Each PCR contained 5 ng genomic DNA in a 10 μL total volume containing MyTaq HS mix (Meridian Bioscience, UK) and 0.2 μM of each primer. Initial specificity testing was conducted using the following amplification conditions: denaturation at 95°C for 3 min; followed by 30 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for 10 s. PCR amplicons were separated through a 1% agarose gel (Meridian Bioscience, UK) containing 0.1 mg/mL ethidium bromide and visualized. A gradient PCR between 52°C and 62°C was conducted to optimize the PCR conditions and confirm the differential amplification of DNA from isolates representing the different groups of strains.

To confirm the loss of mtDNA, DNA from BET-1A ρ0 and BEAS-2B ρ0 cells were compared to the original non-treated source cell line (BET-1A and BEAS-2B) by PCR and gel electrophoresis. PCR reactions were performed in a volume of 25 μl per well on a XYX Technik A6 Thermal Cycler PCR machine with mtDNA tRNA-leu (MT-TL1) and nuclear DNA GAPDH primer sets. Each PCR reaction contained 2 μl annealing primer set, 12.5 μl MyTaq HS mix (Meridian Bioscience, Memphis, TN), 2 μl DNA template (5 ng total), and 8.5 μl PCR water. The PCR conditions were as follows: initial denaturation to 94°C for 1 minute followed by 25 amplification cycles of (94°C for 30 seconds denaturation, ramp down to 55°C 30 secs for annealing, and 72°C for 30 seconds extension) followed by a final 72°C for 5 minutes ramp down to 4°C hold. A 1.5% ultrapure agarose gel in 1X TAE buffer was cast. 10 μl PCR sample and 5 μl 1kb DNA ladder were loaded in each well. Gels were run for 45 mins at 80 volts, and the image was documented with the Bio-RAD ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA USA)