West Nile Virus: Methods and Protocols by Tonya M. Colpitts

By Tonya M. Colpitts

This quantity is a suite of analysis equipment, thoughts, and ways to enquire the molecular biology of West Nile Virus (WNV). Chapters within the publication conceal many elements of WNV, reminiscent of propagation and titration of WNV in vero cells, exam of WNV neuroinvasion and neuropathogenesis within the critical anxious method of a murine version, box surveillance equipment for WNV, and in vitro and in vivo blood-brain barrier types to check WNV pathogenesis. a quick advent, in addition to a dialogue on WNV laboratory security also are integrated. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters contains creation to their respective subject matters, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.

Practical and thorough, West Nile Virus will curiosity virologists, molecular biologists, microbiologists, and different scientists operating with WNV. This e-book is a complete consultant for these accomplishing learn within the box of WNV biology, together with lifestyles cycles, pathogenesis, effect of an infection, and attainable treatment/prevention development.

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2 × 105 cells/ml is usually appropriate for use the following day, while 6 × 104 cells/ml is suitable for a 2-day incubation. 1. 2. Prepare virus inoculum. 001. If virus is from an organ/mosquito homogenate, 1:10 or 1:100 starting dilutions are better, as concentrated tissue material can affect plaque formation. The inoculum volume should be sufficient to cover the monolayer, but no more than about 10 % of that of the usual culture medium volume for the flask being used. 0 ml volumes), depending upon intended use.

Add 1 μL of DNase I (1 U/µL) and incubate at 37 °C for 15 min. 4. Perform a phenol:chloroform extraction using freshly prepared TE-saturated phenol:chloroform. Add 179 μL water and 200 μL phenol:chloroform. Vortex for 15 s and spin at 16,000 × g for 2 min. Carefully remove the top (aqueous) layer and transfer to a fresh microfuge tube. Repeat extraction using chloroform, noting the amount of aqueous phase recovered. 5. Add an equal volume of 5 M ammonium acetate and incubate on ice for 15 min. 6.

Fredericksen Fig. 1 Schematic of workflow. Overview of the steps required to generate recombinant virus from a twoplasmid infectious clone system acquired mutations during amplification of the plasmid in bacteria. Several approaches (see Notes 1–7) have helped to overcome these difficulties [1–19], including the use of a two-plasmid system, which limits the required cloning steps, the use of Gibson Assembly cloning methods or chemical synthesis of portions of the genome, the use of low or very low copy plasmids, and the maintenance of infectious clone plasmids in bacterial strains with low mutation rates [20].

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