"
Team:Osaka/Protocols
From 2012.igem.org
(Difference between revisions)
| (4 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
== Protocols == | == Protocols == | ||
=== Cell survival assay === | === Cell survival assay === | ||
| - | #10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown | + | #10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD<sub>600</sub> reaches 0.4-0.6. |
#Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h. | #Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h. | ||
| - | #Cells were split into 3 ml aliquots in test tubes, and various concentrations of | + | #Cells were split into 3 ml aliquots in test tubes, and various concentrations of DNA damaging agents (we used Mitomycin C and Hydrogen peroxide) were added. |
#After incubation for 2 h with shaking, centrifuge and discard antibacterial agents spiked medium and resuspend with fresh LB medium. | #After incubation for 2 h with shaking, centrifuge and discard antibacterial agents 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). | ||
| Line 16: | Line 16: | ||
=== Promoter assay : Dual-GloR Luciferase Assay System (Promega) === | === Promoter assay : Dual-GloR Luciferase Assay System (Promega) === | ||
| - | #10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown | + | #10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD<sub>600</sub> reaches 0.4-0.6. |
| - | #Cells were split into | + | #Cells were split into 500 µl aliquots in 1.5ml tubes, and various concentrations of DNA damaging agents (we used Mitomycin C and Hydrogen peroxide) were added. |
#After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake. | #After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake. | ||
#Decant supernatant, wash with 1mL PBS | #Decant supernatant, wash with 1mL PBS | ||
| - | #Centrifuge at 3,000g for | + | #Centrifuge at 3,000g for 10 min with soft brake. |
#Decant supernatant, add 100 µl cell lysis buffer. | #Decant supernatant, add 100 µl cell lysis buffer. | ||
#Remove lysate 10-50 µl to the vial. | #Remove lysate 10-50 µl to the vial. | ||
#Add a volume of Dual-GloR Reagent equal to the volume of cell lysate. | #Add a volume of Dual-GloR Reagent equal to the volume of cell lysate. | ||
| - | #Wait at least | + | #Wait at least 10 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer. |
#Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume. | #Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume. | ||
#Wait at least 5 minutes, then measure Renilla luminescence | #Wait at least 5 minutes, then measure Renilla luminescence | ||
#Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter. | #Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter. | ||
Latest revision as of 13:44, 25 September 2012
Protocols
Cell survival assay
- 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD600 reaches 0.4-0.6.
- Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h.
- Cells were split into 3 ml aliquots in test tubes, and various concentrations of DNA damaging agents (we used Mitomycin C and Hydrogen peroxide) were added.
- After incubation for 2 h with shaking, centrifuge and discard antibacterial agents 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 100 µl 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.
Promoter assay : Dual-GloR Luciferase Assay System (Promega)
- 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD600 reaches 0.4-0.6.
- Cells were split into 500 µl aliquots in 1.5ml tubes, and various concentrations of DNA damaging agents (we used Mitomycin C and Hydrogen peroxide) were added.
- After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake.
- Decant supernatant, wash with 1mL PBS
- Centrifuge at 3,000g for 10 min with soft brake.
- Decant supernatant, add 100 µl cell lysis buffer.
- Remove lysate 10-50 µl to the vial.
- Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
- Wait at least 10 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
- Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
- Wait at least 5 minutes, then measure Renilla luminescence
- Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.
