Author: Zirkel, Florian; Kurth, Andreas; Quan, Phenix-Lan; Briese, Thomas; Ellerbrok, Heinz; Pauli, Georg; Leendertz, Fabian H.; Lipkin, W. Ian; Ziebuhr, John; Drosten, Christian; Junglen, Sandra
Title: An Insect Nidovirus Emerging from a Primary Tropical Rainforest Document date: 2011_6_14
ID: ulwo6i38_25
Snippet: For purification, CAVV was harvested by freeze-thawing of infected cells. Cell debris was removed by centrifugation at 3,000 rpm for 20 min, followed by ultracentrifugation through a 36% sucrose cushion at 35,000 rpm (SW40 rotor; Beckman) for 2 h at 4°C. The virus pellet was suspended in 150 l PBS overnight at 4°C. Further purification was achieved on a continuous gradient of 1 to 2 M sucrose in 0.01 M Tris-HCl-4 mM Na-EDTA at 35,000 rpm (SW40 .....
Document: For purification, CAVV was harvested by freeze-thawing of infected cells. Cell debris was removed by centrifugation at 3,000 rpm for 20 min, followed by ultracentrifugation through a 36% sucrose cushion at 35,000 rpm (SW40 rotor; Beckman) for 2 h at 4°C. The virus pellet was suspended in 150 l PBS overnight at 4°C. Further purification was achieved on a continuous gradient of 1 to 2 M sucrose in 0.01 M Tris-HCl-4 mM Na-EDTA at 35,000 rpm (SW40 rotor; Beckman) for 22 h at 4°C. Virus-containing fractions were tested by real-time RT-PCR, and fractions with the highest sequence titers were concentrated through a 36% sucrose cushion at 35,000 rpm (SW40 rotor; Beckman) for 2 h at 4°C. The virus pellet was suspended in 150 l PBS buffer overnight at 4°C.
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