Article Title: A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes
Figure Lengend Snippet: Cloning and characterization of CeHV-2 fosmids. ( a ) Scheme of CeHV-2 genome cloning. Viral DNA was isolated from virus particles, sheared, end-repaired and size fractionated on an agarose gel. Fractionated fragments were ligated into fosmid vector pCC1FOS (red) and packaged into phage lambda particles, which were then used to transduce cells of E. coli strain EPI300. Individual colonies were further characterized by colony PCR, end-sequencing, and restriction digest. ( b ) An overview of all fosmid clones that are characterized by colony PCR and end-sequencing. The genome of CeHV-2 is schematically represented on top, with U S regions drawn on both sides of the U L region. The direction of gene order, in the U L and U S regions, is indicated by arrows and inverted repeats are drawn as boxes. The positions of fosmid inserts are drawn as lines. The names of the fosmids and nucleotide positions of the insertions are given above the respective lines.
Article Snippet: Next-Generation Sequencing and Assembly Library preparation of fosmid-DNA was performed using the NEBnext Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) following the manufacturer’s protocol for < 100 ng DNA input (30–46 ng input amount).
Techniques: Clone Assay, Isolation, Agarose Gel Electrophoresis, Plasmid Preparation, Transduction, Polymerase Chain Reaction, Sequencing