Journal: Virology Journal
Article Title: Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors
Figure Lengend Snippet: Replication of transfected DNA in VAC-infected cells. (A) Southern blot of replicated circular plasmid and linear minichromosome. B-SC-1 cells were infected with VAC and 1 h later transfected with equal molar amounts (20 fmol) of super coiled pUC13 (sc pUC), pUC13 linearized by digestion with EcoR I (lin pUC), linear minichromosome containing pUC13 and 1.3 kbp viral telomeric sequences (lin mc). As a control, cells were mock infected and transfected with 20 fmol of linear minichromosome or 10 times that amount (200 fmol) of super coiled pUC13. At 24 h after infection or mock infection, cells were collected and total DNA extracted. Total DNA (2 μg) was digested with Dpn I subjected to agarose gel electrophoresis and analyzed by Southern blot hybridization using a 32 P-labeled pUC13 probe. Samples (0.5 fmol of lin pUC, 0.5 fmol of lin mc, 1 fmol sc pUC) of the DNA used for transfections (input DNA) were also analyzed. The positions of marker DNA (kbp) are shown on the left. (B) Real-time PCR of replicated plasmid. BS-C-1 cells were transfected with the plasmid p716 at 24 h prior to infection with VAC. At indicated hours post infection (hpi), cells were harvested and total DNA extracted. DNA was untreated or treated with Dpn I or Mbo I and analyzed by real-time PCR using primers specific to plasmid DNA. (C) Quantification of Southern blot. DNA described in panel (B) was digested with EcoR I prior to Mbo I or Dpn I treatment. The digested DNA samples were subjected to gel electrophoresis, transferred to a Nylon membrane, hybridized to a 32 P-labeled p716 probe, and the radioactivity quantified with a phosphoImager.
Article Snippet: Southern blotting DNA (2 μg) was digested with EcoR I and Dpn I or Mbo I, resolved on a 0.8% agarose gel, and transferred to Immobilon-Ny+ (Millipore) transfer membrane.
Techniques: Transfection, Infection, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Hybridization, Labeling, Marker, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Radioactivity