Team:Osaka/Protocols

From 2012.igem.org

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== Protocols ==
== Protocols ==
=== Cell survival assay 1: Mitomycin C ===
=== Cell survival assay 1: Mitomycin C ===
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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 100μl of an overnight culture and grown for 2 h at 37°C.
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#Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
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#Induce parts with IPTG addition to final concentration of 50 µM and incubate for 1h.
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#Add mitomycin C to desired final concentration (we used 2µg/ml) and incubate at 37°C for a further 2h.
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#Cells were split into 3 ml aliquots in test tubes, and various concentrations of antibacterial agents 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 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.
#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.
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=== Cell survival assay 2: Hydrogen peroxide ===
=== Cell survival assay 2: Hydrogen peroxide ===
#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.

Revision as of 07:41, 10 September 2012


Protocols

Cell survival assay 1: Mitomycin C

  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.


Cell survival assay 2: Hydrogen peroxide

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