Vacuum blotter

Southern blot analysis was used to assess telomere length and performed as previously described. Genomic DNA was extracted, digested with XhoI, and then run overnight on a 1% agarose gel at 1 V/cm. Southern transfer was performed using the Biorad Vacuum Blotter according to the manufacturer’s indications. Y′+TG probe labelling and Southern detection were made according to the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche) manufacturer’s instructions. The probe has approximately 1 kb with ~ 880 bp of Y′ and 120 bp of TG repeats, and was cut from pDL1574 using XhoI and BamHI (Dewar and Lydall 2010 (link)).

For Southern blots, genomic DNA was digested to completion overnight with a suitable range of restriction enzymes (New England Biolabs), and separated by agarose gel electrophoresis. For Northern blots total RNA (10 μg) was separated on a 1.2% formaldehyde agarose gel. Genomic DNA digests and total RNA were transferred to positively charged nylon membrane (Amersham Life Science) using a vacuum blotter (Bio-rad, UK). Transferred RNA was fixed to membranes by UVP CL-1000 UV cross-linker (Genetic Research Instrumentation Ltd, Essex, England). Filters were probed with [α-32P] (3,000 Ci/mmol; Amersham, Buckinghamshire, United Kingdom). The labelling of DNAs was performed using Ready-To-Go™ DNA Labelling Beads (Amersham Pharmacia Biotech). Unincorporated [α-³²P]dCTP was removed using ProbeQuant G-50 microspin columns (Amersham Pharmacia Biotech). Hybridization with 32P-labelled probes were carried out at 65 ^oC overnight in Church Buffer (1% BSA, 1 mM EDTA, 0.5 M NaPO4 pH 7.2, 7% SDS). The membranes were rinsed in 2 x SSC (20 x SSC per L: 175.3 g NaCl, 88.2 g sodium citrate) at room temperature, followed by a 20 min wash at 65 ^oC in 2 x SSC and then a wash in 0.5 x SSC at 65 ^oC for 20 min. Damp filters were placed on Kodak X-Omat AR autoradioagraphy film (Sigma) and allowed to expose for the desired time with intensification at −80 ^oC to detect hybridization signals.

Culture was grown overnight in a defined media (-Ura) with glucose. Cells were washed twice in media containing 2% Raffinose and 2% galactose, diluted to OD 0.1 in the same media and grown to an OD of 0.5–0.8 to permit expression of the gal-driven reporters. RNA was isolated using hot phenol extraction followed by two sets of chloroform extraction and ethanol precipitation. 2μg of total RNA was resolved on 1.2% formaldehyde agarose gel, followed by transfer to positively-charged nylon membrane (GE Lifesciences) using a vacuum blotter (Biorad). Next, nucleic acids were UV cross-linked to the membrane and baked at 80°C for 15 minutes. Membranes were then pre-hybridized in Rapid-Hyb buffer (GE Lifesciences) for 30 minutes in a hybridization oven. Radiolabeled DNA probe, which was labeled using polynucleotide kinase and [γ-³²P]ATP, was added to the buffer and incubated overnight. Membranes were washed with nonstringent buffer (2 × SSC, 0.1% SDS) three times, in some cases followed by three washes in stringent buffer (0.2 × SSC, 0.1% SDS), all at hybridization temperature. Membranes were exposed to a phosphorimager screen and analyzed using a Biorad Personal Molecular Imager.
All Northern analyses were performed using at least three biological replicates. Representative images are shown.

Mean telomere length was measured by Southern analysis of telomere terminal restriction fragments (TRF), as previously described [16 (link)]. Genomic DNA samples (5 µg) digested by HinfI were electrophoresed in 0.7 % agarose, 1 × TBE, for 1800 V × H, transferred to a Hybond N⁺ membrane (GE Healthcare) by a vacuum blotter (model 785, Bio-Rad Inc.), hybridized to a 5′ ³²P-labeled telomeric probe, (AACCCT)₃, and exposed to Typhoon FLA 9500 PhosphorImager (GE Healthcare Inc., Hercules, CA, USA). Raw data were analyzed by TeloTool software (corrected mode), specifically designed to obtain average TRF length for each sample [17 (link)]. Sample measurements were reproducibly repeated several times in different gels and averaged.

HBV replicative intermediates from intracellular core particles and HBV transcripts were extracted from transfected cells according to the published protocols18 (link). HBV DNA and RNA were detected by southern/northern blot using PCR-amplified digoxingenin labeled probes (PCR DIG Probe Synthesis Kit, Roche). Briefly, extracted DNA was loaded on 1.2% agarose gel. Total RNA was loaded on formaldehyde denaturing agarose gel. After electrophoresis, DNA/RNA was transferred onto positive charged nylon membrane (Roche) using a vacuum blotter (Bio-rad, USA). Subsequent procedures were performed according to the instruction provided by the DIG High Prime DNA Labeling and Detection Starter Kit II manual (Roche). The levels of HBsAg and HBeAg in culture supernatants were examined by enzyme-linked immunosorbent assay (ELISA) kits (Kehua, Shanghai, China).

Southern blot analysis was used to assess telomere length as previously described [46 (link)]. Genomic DNA was extracted, digested with XhoI and run overnight on a 1% agarose gel at 1 V/cm. Southern transfer was performed using a BioRad Vacuum Blotter according to manufacturer’s instructions. Probe labelling, hybridization and washing were performed according to the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche) instructions. A probe that annealed to the Y’ elements and TG telomere repeats was made using DNA from a plasmid (pDL987) that contained 120 bp of TG repeats and 752 bp of the upstream Y’ element from telomere VIII-R. Loading control pictures were taken from each gel, before transfer, using SYBR Safe to stain the total DNA.