Southern blot analysis.

Cells were harvested at the indicated time points, pelleted, and resuspended in southern lysis buffer (2% SDS, 0.15 M sodium chloride, 10 mM Tris, pH 8, and 1 mM EDTA). The lysed extracts were proteinase K (NEB) treated overnight at 37°C before genomic DNA was sheared with 25-gauge by 5/8-in. 1-mL needle syringe (BD Biosciences). Total DNA content was quantified on a NanoDrop spectrophotometer, and equal amounts of DNA were loaded per well and electrophoresed on a 1% agarose gel at 33 V overnight. DNA was transferred to a nitrocellulose membrane and hybridized with homologous genomic clones of MVMp. The MVMp probe was generated by agarose gel extraction and purification (Qiagen) of BamHI-XbaI double-digested MVMp infectious clone.