Notebook:Jason R. Kelly/2008/02/13
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|iGEM Project name 1||Main project page|
FACS protocol V0.1
- 5 strains from Jason Kelly
- TOP10 background strain (Jason will provide so we're all actually using the same one).
- 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS)
- 5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235
Live Cell Procedure
- When your 5 mL culture reaches OD600 = 0.100, pellet the cells by centrifuging at 3000xg at room temperature for 5 minutes.
- Remove the supernatant.
- Resuspend the cell pellet in 5 mL PBS.
- Re-pellet the cells by centrifuging at 3000xg at room temp for 5 min.
- Repeat wash with 5 mL PBS.
- Re-suspend washed cells in 0.5 mL PBS. The cell concentration should be approximately 5x108 cells/mL.
- Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
- Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
- Put cells on ice.
- Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.