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Genetic Markers to Isolate Recombinants1711.Humidified incubator, 3712 Genetic Markers to Isolate Recombinants1711.Humidified incubator, 3712

Genetic Markers to Isolate Recombinants1711.Humidified incubator, 3712 - PDF document

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Genetic Markers to Isolate Recombinants1711.Humidified incubator, 3712 - PPT Presentation

3904 303 PM17 Genetic Markers to Isolate Recombinants19 Fig 119 3904 303 PM19 20Lorenzo Galindo and Blasco1About 24 h before infection split 13 a confluent culture of incubator for 202 ID: 411525

3/9/04 3:03 PM17 Genetic Markers

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Genetic Markers to Isolate Recombinants1711.Humidified incubator, 3712.Disposable sterile scraper.13.5 mL Polypropylene tubes.14.5 mL Polystyrene tubes.15.Complete 2X EMEM medium: 2X EMEM, supplemented with 4 m glutamine,16.2% low-melting point (LMP) agarose in water, autoclaved.17.4% X-gal in dimethylformamide.18.10 mg/mL neutral red in water.19.Sterile Pasteur pipets.20.10 mg/mL MPA in 0.1 21.10 mg/mL xanthine in 0.1 22.10 mg/mL hypoxanthine in water.23.GPT selection medium: complete EMEM containing 1/400 vol of 10 mg/mL24.4525.5 mg/mL crystal violet staining solution.In the plasmid, the F13L coding sequence is flanked by vaccinia virus DNA selection is based on the incorporation in the vaccinia genome of gene and its expression under the control of vaccinia virus expression confers resistance to MPA, an inhibitor of the can be isolated in a selec- 3/9/04, 3:03 PM17 Genetic Markers to Isolate Recombinants19 Fig. 1.19 3/9/04, 3:03 PM19 20Lorenzo, Galindo, and Blasco1.About 24 h before infection, split 1:3 a confluent culture of incubator for 202.Prepare virus inoculum (1 mL/well) by diluting a crude virus stock (3.Remove cell growth medium from the cultures and immediately add virus inocu-4.About 45 min before transfection, prepare calcium phosphate precipitate as fol- CaCl droplet by droplet,5.Aspirate virus inoculum from the CV-1 cell cultures, and add the precipitate on6.Add 2 mL per well of cell growth medium. Incubate 4 h in a CO incubator7.Remove medium from the cultures, and replace with 2 mL fresh cell growth8.Harvest the infected cells from the well with a disposable rubber scraper or by9.Release progeny virus from cells by repeated freezeThe cell lysate can be stored at 3/9/04, 3:03 PM20 22Lorenzo, Galindo, and Blasco or a control plasmid not carrying gene F13L 3/9/04, 3:03 PM22 Genetic Markers to Isolate Recombinants235.Aspirate off growth medium from the BSC-1 cell monolayers and add 1 mL of6.Melt sterile 2% LMP agarose in water by heating in a microwave oven and cool7.Immediately before use, prepare EMEM8.Remove virus inoculum from each well and add 3 mL EMEM9.Stain the monolayer by overlaying the EMEM agarose with the addition of 2 mL10.When plaques are clearly visible, pick well-separated plaques by plunging a ster-Note 12ing 0.5 mL 11.Repeat 1.Seed BSC-1 cells in six-well tissue culture plates and incubate until the cells are2.Twelve to twenty-four hours before infection, prepare GPT selection medium.3.Thaw the cell lysate obtained at the completion of , remove0.5 mL, 4.Sonicate in an ice/water bath sonicator for at least three cycles of 15 s until the5.Make 10-fold serial dilutions of the infection/transfection cell lysate in cell 02/Lorenzo/15-303/9/04, 3:03 PM23