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Team:Freiburg Bioware/Project/Methods
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| + | =Method Development= | ||
| + | ==Serum-free cell culture medium== | ||
| + | ===Introduction=== | ||
| + | Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum). | ||
| + | The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. | ||
| + | Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients. | ||
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Revision as of 17:26, 27 October 2010
Methods
Contents |
Method Development
Serum-free cell culture medium
Introduction
Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum). The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium. Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.
https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/ITRDiary
Protocols; Standard Operating Procedures
Standard Protocol: Cloning
Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf
Media:Split_cellculture.pdf
Media:Freeze_cellculture.pdf
Media:production of competent E.coli.pdf
Media:Freiburg10_Transfection_protocoll.pdf
Media:Freiburg10 Thawing cells.pdf
Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf
Media:Freiburg10 Endotoxinfreie Midi.pdf
Media:Freiburg10_LB+Agar.pdf
Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf
Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf
