Dig high prime dna labelling and detection starter kit

The DIG-High Prime DNA Labelling and Detection Starter Kit (11585614910, Roche, Germany) was used in the experiments. The HBV core DNA and Hirt-extracted DNA samples were subjected to 1% agarose gel electrophoresis and transferred onto a nylon membrane. The nylon membrane was cross-linked by exposure to UV light in a Stratalinker UV crosslinker and hybridised with a digoxigenin-labelled HBV full-length genomic DNA probe overnight at 42°C. The membrane was incubated in blocking solution for 30 min and antibody solution for another 30 min at 37°C. The signal was detected by exposing on an X-ray film. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA.
For HBV DNA probe, full-length HBV DNA was amplified by PCR using pCH9/3091 plasmid as template. Electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit (D2111-02, Magen, China) and PCR product purification kit (11732676001, Roche, Germany). Full-length HBV DNA was labelled with digoxigenin-11-dUTP with a kit supplied by Roche (11585614910). Labelling was carried out by following the manufacturers’ instructions.

Genomic DNA digested with EcoRI was electrophoresed on a 1.5% agarose gel and transferred onto a positively charged membrane for Southern blotting analyses (Bio-Rad, Hercules, Calif., USA). The clones of interest were amplified and labelled with digoxigenin-dUTP and used as probes for hybridization, which was performed overnight at 47°C for clones Ac4p1 and Ac19p1 and at 46°C for clone Ac4p3. Stringency washes and detection of signals were performed following the DIG High Prime DNA labelling and Detection Starter Kit (Roche, Indianapolis, IN) manufacturer’s instructions. The probes used for Southern blotting were also used in dot blot analyses. For qualitative analyses, 100 ng of each genomic DNA was spotted and fixed (120°C, 30 min) onto a positively charged membrane. For quantitative dot blot analyses, 1 ng, 0.5 ng, 0.25 ng, 0.12 ng, 0.06 ng, and 0.03 ng of genomic DNA were used. Hybridization and detection were carried out as specified for Southern blotting.

Transconjugants were further confirmed by Southern blotting. SmaI- and S1-PFGE analyses were performed as described previously (Tomita et al., 2002 (link); Yan et al., 2011 (link)). Southern blotting was performed using a DIG High Prime DNA labelling and Detection Starter Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The digoxigenin-labelled lsa(E)-specific probe was prepared using primers (forward 5′-ACAGCGAGTTGTTTCCTGCT-3′; and reverse 5′-GCACGTTTCATCGCTTTTGC-3′) that amplified a 410-bp region of the lsa(E) gene. After S1-PFGE, the DNA was transferred to a nylon membrane (Hybond N, Amersham, United Kingdom) that was hybridised with the prepared lsa(E)-specific probe. Detection was performed using an NBT/BCIP colour detection kit (Roche, Switzerland).