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: Mitomycin C and Hydrogen peroxide ===
#10 ml of LB broth was inoculated with 100μl of an overnight culture and grown for 2 h at 37°C.
#10 ml of LB broth was inoculated with 100μl of an overnight culture and grown for 2 h at 37°C.
#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.
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=== Cell survival assay 2: Hydrogen peroxide ===
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=== Promoter assay : Dual-GloR Luciferase Assay System (Promega) ===
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#Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
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#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 5 min with soft brake.
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#Decant supernatant, add 100 ul cell lysis buffer.
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#Remove lysate 10-50 ul 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 5 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.
 +
#Wait at least 5 minutes, then measure Renilla luminescence
 +
#Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.

Revision as of 07:45, 10 September 2012


Protocols

Cell survival assay: Mitomycin C and Hydrogen peroxide

  1. 10 ml of LB broth was inoculated with 100μl of an overnight culture and grown for 2 h at 37°C.
  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 antibacterial agents 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. Centrifuge the incubative tube at 3,000g for 15 min with soft brake.
  2. Decant supernatant, wash with 1mL PBS
  3. Centrifuge at 3,000g for 5 min with soft brake.
  4. Decant supernatant, add 100 ul cell lysis buffer.
  5. Remove lysate 10-50 ul to the vial.
  6. Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
  7. Wait at least 5 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
  8. Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
  9. Wait at least 5 minutes, then measure Renilla luminescence
  10. Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.
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