Lab9-2011

Lab9-2011 - BIOL 513 Lab 9. Detection of viral antigens Lab...

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BIOL 513 – Lab 9. Detection of viral antigens 144 Lab 9: Detection of viral antigens - lab designed for 12 groups of 2 students Equipment for Lab 9: Day 1 Day 2 Day 3 Day 4 Day 5 CO 2 incubator + - - - -20°C freezer + - - - Inverted phase contrast microscope + + - - Bench-top microcentrifuge + - - - 2-liter vacuum flask with aspirator 2 2 - - Power supplies - - 6 - Protein gel electrophoresis apparatus - - 6 - Protein electroblotting apparatus - - 6 - Rocker - - 2 2 Safety bulb 12 12 12 - Pipetman, 40-200- µ L - 12 12 - Pipetman, 100-1000- µ L 12 - - - Vortex - - 6 - Timers - - 6 - 500-mL cylinders - - 6 - 1000-mL cylinders - - 6 - Plastic trays (see details) - - 6 6 Forceps - - 12 12 250-mL beaker 12 - - - Rack for microcentrifuge tubes 12 12 12 - Rack for 15-mL tubes 12 12 - - Rack for 50-mL tubes 12 - - - Heat block- 98°C - - + - Supplies & Consumables for Lab 9: Day 1 Day 2 Day 3 Day 4 Day 5 12% SDS-PAGE gel - - 6 - Microcentrifuge tubes 36 - + - 1-mL pipet 48 24 - - 10-mL pipets - - 24 6 Sterile Pasteur pipet 48 - - - Yellow pipet tips in trays 12 - + - Gel loading pipet tips - - + - Blue pipet tips in trays 12 - - - SDS-PAGE sample buffer - - 5 mL - SDS-PAGE running buffer - - 5 L - BLOTTO (see details) - - 200 mL - Western wash buffer - - - + Whatman 3MM - - + - PBS + + + + Distilled water + + + - Methanol + + + - 70% ethanol in spray bottle 6 6 6 6 Paper towels - - - + 1% methylcellulose overlay 60 mL Western transfer buffer - - 3 L - - 1% BSA in PBS - 8 mL - - - 35-mm dishes 72 - - - -
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BIOL 513 – Lab 9. Detection of viral antigens 145 Lab 9: Detection of viral antigens - lab designed for 12 groups of 2 students Materials for Lab 9 provided by the instructor: Instructors: On Sunday, you will need to plate CrFk cells for Monday (see below for details). Day 1, Monday, April 18, 2011 (Note to students and instructors: on this day, students will need to come back to the lab several hours after the lab period has finished, at a point when Haworth is locked. Be sure to make the proper arrangements.) CrFk cells, a set of 6 × 35-mm dishes @ ~100% confluency – 12 sets (seeded the day before from 3 × 150-mm TC dishes @2 × 10 7 cells/dish need 72 × 10 6 total cells to seed each 35 mm dish with 1 × 10 6 cells) FCV, 1 mL @ 500 PFU/mL, in M199, 2%FCS, 1 × AA, in a 1.5-mL tube – 12 1% methylcellulose overlay, 5 mL in a 15-mL tube – 12 Methanol, 4 mL in a 15-mL tube – 12 CrFk growth medium (DMEM +10% FCS, 1 × AA), 20 mL in a 50-mL tube – 12 PBS, 10 mL in a 15-mL tube – 12 Day 2, Tuesday, April 19, 2011 Anti-FCV immune serum (hyperimmune mouse aceties fluid - HMAF), 1:250 in PBS+1%BSA, 0.6 mL in a 1.5 mL tube – 12 Goat anti-mouse peroxidase conjugate, 1:5000 in PBS+1%BSA, 0.6 mL in a 1.5-mL tube – 12 PBS+1%BSA, 30 mL in a 50-mL conical tube – 12
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This note was uploaded on 01/22/2011 for the course BIO 513 taught by Professor David during the Spring '10 term at Kansas.

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Lab9-2011 - BIOL 513 Lab 9. Detection of viral antigens Lab...

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