Taq pcr master mix 2

Eight pairs of primers (Table 4) were designed to verify regions of the DSLV1 genome where the coverage of reads was low or ambiguous. PCR reactions (25 μL) contained 0.4 mM forward and reverse primers (Table 4), 12.5 μL Taq PCR master mix 2 × (Sangon Biotech), and 20–25 ng of genomic DNA. The PCR cycle conditions were as follows: initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 54°C (primers 1–4 and 6–8) or 56°C (primer 5) for 30 s, and 72°C for 45 s. Sequences obtained by PCR and sequencing (Sangon Biotech) were then aligned with the assembled genomes to confirm the accuracy of the sequence assembly.

PCR reactions (25 μL) contained 0.4 mM forward and reverse primers (Table 2), 12.5 μL Taq PCR master mix 2 × (Sangon Biotech), and 20–25 ng of genomic DNA. The PCR cycle conditions are given in Table 3. After agarose gel electrophoresis and ethidium bromide staining, PCR amplicons were visualized with a Gel Doc XR+ system (Bio-Rad). PCR products were purified from the gel using a universal DNA purification kit (Tiangen Biotech), then ligated into the pUCm-T vector (Sangon Biotech). Recombinant plasmids were transformed into TOP10 cells (Tiangen Biotech), which were streaked on agar plates containing 50 μg/mL ampicillin and grown overnight at 37°C. Three positive colonies were selected for Sanger sequencing (Sangon Biotech).