Amersham hybond n

DNA purified from chromatin imunoprecipitated with the anti CENP-A antibody [9 (link), 21 (link)] and input DNA were transferred to nylon membranes (Amersham HybondTM-N, GE Healthcare) through a Minifold II apparatus (Schleicher and Schuell) and denatured. The membranes were hybridized at 64 °C for 18 h in Church buffer containing one of the following ³²P-α[dCTP]-labelled probes, generated by random primer labelling: a 7 kb EcoRI/SacI 37cen fragment and a 7.2 kb EcoRI/SacI 2PI fragment [10 (link)]; a 441 bp PCR-amplified fragment from horse genomic DNA, containing an ERE-1 insertion [39 (link)].
After hybridization, the membranes were washed twice in 2× SSC, 0.5 % SDS for 15 min at 64 °C and once in 0.2× SSC, 0.5 % SDS for 30 min at 64 °C. Radioactive signals were detected using a phosphorimager (Cyclone, Packard) and the densitometric analysis was performed with the ImageJ 1.48v software [31 (link)].

The full-length HBV DNA fragment (~3.2 kb) was prepared from pBR-3HBneo [33 (link)] with BamHI digestion followed by gel-purification and was used as a probe to detect HBV DNA on the Southern blot. The DNA was denatured at 100 °C for 5 min and labeled using Amersham^TM AlkPhos Direct Labelling Reagents according to the manufacturer’s protocols (Cat#RPN3680; GE Healthcare).
The extracted intracellular core-associated and extracellular virion-associated and HBV DNA from the transfected cells and medium, respectively, were run on 1.2% TAE agarose gel. After the electrophoresis, the gel was treated with 0.2N HCl for 10 min, 0.5N NaOH-0.6M NaCl for 20 min and 0.5M Tris-HCl (pH 8.0)-1.5M NaCl for 10 min. The DNA in gel was transferred onto a nylon membrane (Amersham Hybond^TM-N⁺; GE Healthcare) through 0.4N NaOH overnight. The membrane was then cross-linked with UV and washed with 3 x SSC (saline sodium citrate; 20 x SSC, 3 M NaCl-0.3 M 3Na-citrate, pH 7.0) and hybridized with the probe at 55 °C overnight. Then, the membrane was washed with a washing buffer (urea 12%, SDS 0.1%, 50 mM NaHP₂, 150 mM NaCl, 1.0 mM MgCl₂ and 0.002% blocking reagent), washed with a detection buffer (50 mM NaCl, 2 mM MgCl₂ in DWB [1.0 M Tris, pH 10]) and rinsed with CDP-STAR (Cat# 12041677001, Roche) for 5 min for chemiluminescence, which was detected and visualized by a ChemiDoc™ imaging system (Bio-Rad).