The BioBricks Foundation:Standards/Technical/Measurement/Promoter characterization experiment (FACS)
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FACS protocol V0.1
Materials
- 5 promoter test strains provided as stabs from Endy Lab
- <bbpart>I20259</bbpart>,<bbpart>I20260</bbpart>,<bbpart>I20268</bbpart>,<bbpart>I20269</bbpart>,<bbpart>I20270</bbpart>
- 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
- Jason R. Kelly 13:37, 15 February 2008 (EST):What's the cell strainer cap for? Don't normally use that.
Culturing of the strains
- Streak LB+Kan plates of the 5 test strains and an LB (no antibiotic) for TOP10
- For each test construct add 5ml of supplemented M9 medium (w/glycerol, see preparation protocol) and Kanamycin (20ug/ml) to X 17mm test tubes. Add 5ml of supplemented M9 medium (w/glycerol) with no antibiotic for the TOP10 negative control cells to X 17mm test tubes.
- how many replicates do we want to do (X)?
- Inoculate media by picking single colonies from plates. Grow cultures for 20 hrs at 37°C with spinning at 70 rpm.
- In the morning dilute 100x into 5ml of pre-warmed (37C) fresh media to get cells back into log phase and let grow for 4hrs under same conditions as overnight
- After four hours, a 500ul aliquot from each culture is used for OD600 measurement. The cultures should then be diluted to OD 0.07 in 5 ml of pre-warmed fresh media to be grown for one hour under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
- In our experience the results are the same w/o this second dilution (e.g. just taking measurements after the 4 hour step)
- After 1hr the cultures should be at an OD of 0.1 - 0.15, take cells and put immediately on ice.
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.