Team:Osaka/Protocols

From 2012.igem.org

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== Protocols ==
== Protocols ==
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=== Cell survival assay 1: UV irradiation ===
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=== Cell survival assay 2: 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.
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#Induce parts with IPTG addition (to final concentration of 100µM) for 1h.
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#Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
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#Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive).
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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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#Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
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#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
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#Irradiate cells on the agar with UV light at desired energy dosage.
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#[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 50μl 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.
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#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.
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#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.

Revision as of 13:28, 28 August 2012


Protocols

Cell survival assay 2: Mitomycin C

  1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
  2. Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
  3. Add mitomycin C to desired final concentration (we used 2µg/ml) and incubate at 37°C for a further 2h.
  4. Centrifuge, discard mitomycin C-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 50μl 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.
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