1. Propagation of virus
- Start with 225 cm2 flask or three 10-cm dishes.
- Infect host cells with virus.
- Incubate cultured cells for 2 to 3 days at 37oC.
- Pellet infected cells.
Tips: You may store infected cells at -80oC at this step.
2. Purification of virus
- Suspend cells in 10 ml of sonication buffer.
- Sonicate or freeze-and-thaw the virus suspension.
- Centrifuge at 9,400 g for 10 min at 4oC.
- Filtrate supernatant through 0.2-um polyethersulfone filter unit (Millipore).
- Concentrate flowthrough into 1 ml by tangental flow filtration with Amicon Ultra-15 MWCO 100K filter (Millipore) at 5,000 g for 15 min at 4oC.
- Spin at 604,000 g for 15 min at 4oC by ultracentrifugation on a CsCl gradient.
- Recover 1 ml of virus sample.
Tips: Use 5 ml seal tube. Layer virus solution (ca. 1 ml), 2.2M CsCl (3 ml), and 4.0M CsCl (1 ml) in this order.
3. Removal of CsCl
- Add 9 ml of displacement buffer.
- Concentrate flowthrough into 1 ml by tangental flow filtration with Amicon Ultra-15 MWCO 100K filter (Millipore) at 2,500 g for 5 min at 4oC.
- Add 9 ml of displacement buffer directly to filtration unit.
- Concentrate flowthrough into 1 ml by tangental flow filtration with Amicon Ultra-15 MWCO 100K filter (Millipore) at 2,500 g for 5 min at 4oC.
- Transfer virus solution to a new tube.
Tips: Suspend well before following centrifugation.
4. Reagent
sonication buffer (1000 ml)
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10 mM HEPES, pH 8.0 (10 ml/1 M)
2.2M CsCl (100 ml)
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2.2 M CsCl (38 g)
10 mM HEPES, pH 8.0 (1 ml/1 M)
4.0M CsCl (100 ml)
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4.0 M CsCl (67 g)
10 mM HEPES, pH 8.0 (1 ml/1 M)
displacement buffer (1000 ml)
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10 mM HEPES, pH 8.0 (10 ml/1 M)
2 mM MgCl2 (10 ml/0.2 M)
10% glycerol (100 ml)
4% sucrose (40 g)