Team:Osaka/Protocols

From 2012.igem.org

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== Protocols ==
== Protocols ==
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=== Cell survival assay 1: Mitomycin C ===
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=== Cell survival assay ===
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#Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
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#10 ml of LB broth was inoculated with 100 &micro;l of an overnight culture and grown until the OD<sub>600</sub> reaches 0.4-0.6.
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#Induce parts with IPTG addition to final concentration of 100&micro;M and incubate for 1h.
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#Induce parts with IPTG addition to final concentration of 50 &micro;M and incubate for 1h.
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#Add mitomycin C to desired final concentration (we used 2&micro;g/ml) and incubate at 37&deg;C for a further 2h.
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#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.
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#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
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#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).
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#Pipette 100μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.  
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#Pipette 100 &micro;l to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.  
#Wrap plates in aluminium foil and incubate at 37&deg;C.
#Wrap plates in aluminium foil and incubate at 37&deg;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.
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=== Cell survival assay 2: Hydrogen peroxide ===
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#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.
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=== Promoter assay : Dual-GloR Luciferase Assay System (Promega) ===
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#10 ml of LB broth was inoculated with 100 &micro;l of an overnight culture and grown until the OD<sub>600</sub> reaches 0.4-0.6.
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#Cells were split into 500 &micro;l aliquots in 1.5ml tubes, and various concentrations of DNA damaging agents (we used Mitomycin C and Hydrogen peroxide) were added.
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#After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake.
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#Decant supernatant, wash with 1mL PBS
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#Centrifuge at 3,000g for 10 min with soft brake.
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#Decant supernatant, add 100 &micro;l cell lysis buffer.
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#Remove lysate 10-50 &micro;l to the vial.
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#Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
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#Wait at least 10 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
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#Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
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#Wait at least 5 minutes, then measure Renilla luminescence
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#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

  1. 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD600 reaches 0.4-0.6.
  2. Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h.
  3. 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.
  4. After incubation for 2 h with shaking, centrifuge and discard antibacterial agents 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 100 µl 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.


Promoter assay : Dual-GloR Luciferase Assay System (Promega)

  1. 10 ml of LB broth was inoculated with 100 µl of an overnight culture and grown until the OD600 reaches 0.4-0.6.
  2. 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.
  3. After incubation for 2 h with shaking, centrifuge the incubative tube at 3,000g for 15 min with soft brake.
  4. Decant supernatant, wash with 1mL PBS
  5. Centrifuge at 3,000g for 10 min with soft brake.
  6. Decant supernatant, add 100 µl cell lysis buffer.
  7. Remove lysate 10-50 µl to the vial.
  8. Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
  9. Wait at least 10 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
  10. Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
  11. Wait at least 5 minutes, then measure Renilla luminescence
  12. Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.
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