Team:Osaka/Protocols
From 2012.igem.org
(Difference between revisions)
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== Protocols == | == Protocols == | ||
- | === Cell survival assay | + | === Cell survival assay 1: Mitomycin C === |
#Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h. | #Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h. | ||
#Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h. | #Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h. | ||
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#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium. | #Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium. | ||
#[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). | #[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). | ||
- | #Pipette | + | #Pipette 100μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. |
#Wrap plates in aluminium foil and incubate at 37°C. | #Wrap plates in aluminium foil and incubate at 37°C. | ||
#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates. | #After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates. | ||
+ | |||
+ | |||
+ | === Cell survival assay 2: Hydrogen peroxide water === | ||
+ | #Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h. |
Revision as of 13:33, 28 August 2012
Protocols
Cell survival assay 1: Mitomycin C
- Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
- Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
- Add mitomycin C to desired final concentration (we used 2µg/ml) and incubate at 37°C for a further 2h.
- Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
- [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).
- Pipette 100μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
- Wrap plates in aluminium foil and incubate at 37°C.
- After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.
Cell survival assay 2: Hydrogen peroxide water
- Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.