Team:Osaka/Protocols

Protocols

Cell survival assay 1: Mitomycin C

1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
2. Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
3. Add mitomycin C to desired final concentration (we used 2µg/ml) and incubate at 37°C for a further 2h.
4. Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
5. [RECOMMENDED] Dilute pre-culture samples according to expected survival rate, as determined from a preliminary experiment (lower dilution rate is needed if fewer cells are expected to survive).
6. Pipette 100μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
7. Wrap plates in aluminium foil and incubate at 37°C.
8. After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.

Cell survival assay 2: Hydrogen peroxide

1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.