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> 4.Experimental Procedures |
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About this Database
> 4.Experimental Procedures |
| Last Updated (October 15, 2004) |
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About this Database Toxoplasma Full-Length cDNA project 2.Construction of Toxoplasma Full-Length cDNA Library I) Cultivation of Toxoplasma parasites The RH strain of Toxoplasma gondii tachyzoites (1x107 parasites) were infected to Vero (T25) cells and incubated in EMEM medium supplemented with 8% fetal bovine serum for 72 hours in vitro. Infected cells were collected with a scraper and passed through 27G needle for three times and purified by filtration through Millex SV filters (pore size 5 um, Millipore). Cells were collected by centrifugation at 2,000 rpm for 10 min and washed in EMEM medium without FBS. Parasites were suspended at 1x107/ml in EMEM medium without FBS. Five-week old ICR female mice were infected with 1 ml parasites i.p.. Three days after infection, mice were sacrificed by cervical dislocation. Parasites were collected by washing the peritoneal cavity with 5 ml EMEM, passed through 5 um filter, washed in PBS and suspended at 2-3x109 parasite/ml. 20 ml TRI REGENT (Sigma) was added to 1 ml parasite solution, mixed by pipetting, frozen in liquid nitrogen and kept at -80C until used. II) Production of a full-length cDNA A full-length enriched library was produced by oligo-capping method as described in Full-malaria. |
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