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.

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).
- Jason R. Kelly 14:14, 15 February 2008 (EST):In our experience the results are the same w/o this second dilution (e.g. just taking measurements after the 4 hour step). So if it's too onerous we can probably just skip this.
After 1hr the cultures should be at an OD of 0.1 - 0.15.

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.