Lab3-2011

Lab3-2011 - BIOL 513 Lab 3 Virus characterization Part 1...

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BIOL 513 – Lab 3: Virus characterization – Part 1. Evaluation of virion size 53 Lab 3: Virus characterization – Part 1. Evaluation of virion size - lab designed for 12 groups Equipment for Lab 3: Day 1 Day 2 Day 3 Day 4 Day 5 CO 2 incubator + + 6-ft biosafety cabinet - 2 Light microscope - + Inverted phase contrast microscope + - Hemocytometer with coverslip - 12 Hand counter - 12 Vortex 6 - 2-liter vacuum flask with aspirator 2 2 Pipetman, 40-200- µ L 12 12 8-channel Pipetman, 50-300 µ L - 6 Safety bulb 24 12 250-mL beaker 12 - Forceps - 12 Rack for microcentrifuge tubes - 12 Rack for 12x75 mm tubes 12 - Rack for 15-mL tubes - 12 Rack for 50-mL tubes 12 - Supplies & Consumables for Lab 3: Day 1 Day 2 Day 3 Day 4 Day 5 96-well tissue culture plate cluster - 12 15-mL sterile conical tube - - Sterile 12x75-mm tube with snap cap 576 - Microcentrufuge tube - 12 Sterile dilution trays - 24 1-mL pipet 24 - 5-mL pipet 60 - 10-mL pipet - 26 Sterile Pasteur pipet 12 6 0.45- µ m sterile filter 24 - 0.10- µ m sterile filter 24 - 3-mL sterile syringe with luer-lock 48 - Sterile filtered pipet tips in trays 12 12 0.4% Trypan blue stain - + Crystal violet stain - + 70% ethanol in a spray bottle 6 6 Paper towels - + KimWipes + + 50-mL sterile conical tube 12 -
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BIOL 513 – Lab 3: Virus characterization – Part 1. Evaluation of virion size 54 Lab 3: Virus characterization – Part 1. Evaluation of virion size - lab designed for 12 groups Materials for Lab 3 provided by the instructor: Day 1: Tuesday, February 15, 2011 Number-encoded FCV and FHV suspensions, a set of 3x1 mL each, both at 5x10 6 PFU/mL – 12 sets 15 mL of 2% BSA in PBS filtered through a 0.22- µ m filter – 12 50-mL tube with 50 mL CrFk cell culture medium (DMEM +10% FCS, 1x AA) – 12 Extra 96-well plate cluster with seeded cells – 6 Extra CrFk medium Day 2, Monday, February 21, 2011 12 mL suspension of freshly trypsinized CrFk cells @ 4x10 5 cell/mL – 12 (from 3 150- mm plates @ ~2x10 7 cells/plate to get total 60x10 6 cells) Extra CrFk medium
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BIOL 513 – Lab 3: Virus characterization – Part 1. Evaluation of virion size 55 LAB 3 INTRODUCTION TO VIRAL CHARACTERIZATION Estimation of Viral Size Size is a useful parameter in the classification of viruses. Size can be determined directly by electron microscopy or indirectly by using membranes with varying pore sizes. When using membranes, the cell debris is removed from the viral suspension by centrifugation and other smaller particulate matter is removed by filtering the viral suspension through a 300-nm filter. The resulting viral suspension is then passed through a 100-nm and a 50-nm filter.
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Lab3-2011 - BIOL 513 Lab 3 Virus characterization Part 1...

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