A gblock gene fragment

A gBlock Gene Fragment (Integrated DNA Technologies, Coralville, IO, USA) was designed containing the amplicons for the four phage species, Erwinia amylovora, and Pantoea agglomerans listed in Table 4. The ends each contain an EcoRI restriction site with an additional external 10 random bases to aid EcoRI digestion. The fragment was cloned into a pIDTBlue vector modified with an inserted EcoRI site to create the plasmid pTotalStdA (Figure 1). The construct was transformed into TOP10 Chemically Competent E. coli cells (Life Technologies, Carlsbad, CA, USA). The transformed cells were grown in 2% LB broth (BD, Sparks, MD, USA) with 200 µg/mL ampicillin overnight at 37 °C, 200 rpm. Plasmid isolation was carried out using the Qiagen Plasmid Mini Kit (Qiagen, Toronto, ON, Canada) as per manufacturer’s instructions. The plasmid was linearized using ScaI restriction enzyme (New England Biolabs, Ipswich, MA, USA), quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and diluted to 10¹² copies/mL in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA). A log₁₀ dilution series was prepared in TE and analyzed with qPCR for all six primer and probe sets to ensure proper dilution and amplification.