Team:Heidelberg/Notebook/Methods
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Laura Nadine (Talk | contribs) (→Virus Production) |
Laura Nadine (Talk | contribs) (→Virus Production) |
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:'''Seeding''' | :'''Seeding''' | ||
- | Virus production was done in 150cm< | + | Virus production was done in 150cm<sup>2</sup> flasks. 0.9 million cells were seeded in 30ml medium per flask. Transfections were done as described [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection above]. |
:'''Harvesting''' | :'''Harvesting''' | ||
To harvest the cells, cell suspension was decanted into 500ml corning conical centrifuge tubes. Remaining cells were washed with 19ml PBS and also transferred into the tubes. Cells were collected by centrifugation at 1500rpm for 15min at 4°C. Supernatant was aspirated, cells resuspended in 10ml 1xPBS and transferred into a 50ml blue-cap vial. Centrifugaion as above, aspiration of supernatant. Pellet was then resuspended in 5ml virus lysate solution and frozen at -196°C in liquid nitrogen for 5min, then thawed at 37°C. This freeze/thaw cycle was repeated five times. | To harvest the cells, cell suspension was decanted into 500ml corning conical centrifuge tubes. Remaining cells were washed with 19ml PBS and also transferred into the tubes. Cells were collected by centrifugation at 1500rpm for 15min at 4°C. Supernatant was aspirated, cells resuspended in 10ml 1xPBS and transferred into a 50ml blue-cap vial. Centrifugaion as above, aspiration of supernatant. Pellet was then resuspended in 5ml virus lysate solution and frozen at -196°C in liquid nitrogen for 5min, then thawed at 37°C. This freeze/thaw cycle was repeated five times. |
Revision as of 14:40, 25 October 2010
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