Dig high prime dna labeling and detection starter kit

Before Southern or Northern blotting, cytoplasmic viral core particles were isolated from Huh7 cells 48h post-transfection as described previously26 (link). The isolated core particles were incubated with 50 U micrococcal Nuclease S7 (Fermentas) to remove the nonencapsidated viral DNA and RNA completely. Core-associated DNA and RNA were extracted from each aliquot of core particles, respectively, as described previously27 (link). For Southern blot determination of newly synthesized viral genome, extracted core-associated DNAs were treated with DpnI to remove input rcccDNA. The resulted DNAs and RNAs were separated by agarose gel electrophoresis as described in27 (link), and hybridized to a DIG-labeled random-primed probe specific for the HBV sequence. Finally, the viral DNA and RNA bands were illustrated by a horseradish peroxidase-labeled anti-digoxigenin antibody using the DIG-High Prime DNA Labeling and Detection Starter Kit (Roche China, Shanghai).
The digoxigenin (DIG)-labeled probes for Southern and Northern blot were synthesized using the DIG-High Prime DNA Labeling and Detection Starter Kit (Roche), according to the manufacturer’s instructions.

The transformants and the WT strains of C. magnum were transferred to 100 mL fresh liquid PDA and cultured for 20 d at 27°C in the dark. Genomic DNA of 40 transformants and the WT strains of C. magnum were extracted by CTAB (Sangon Biotech, China).
To analyze genome integration of the transferred genes, the hph gene was amplified using the primer pair hph-F + hph-R (Table 1) and sequenced. PCR was were performed according to the protocol described by Guo et al. with slight modifications (Guo et al., 2022 (link)). The annealing temperature of hph was 60°C. PCR amplicons were purified and sequenced by Sangon Biotech Company (Shanghai, China).
The digestion of genomic DNA from 40 transformants or WT strains with EcoRI and HindIII (NEB, Ipswich, United States), electrophoresised on 0.75% agarose, were depurated, and transferred to nylon membranes (Hybond-N+; General Electric Company, Boston, United States). In the hybridization experiments, the digoxigenin (DIG) labeled probe corresponded to 1,380 bp hph of T-DNA from plasmid pATMT1 (Figure 1), and the hph gene was amplified using the primers hph-F and hph-R. Hybridization and chemiluminescence detection of the hybridized Dig-labeled probes were performed using the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Germany).