Team:Osaka/Protocols

Cell survival assay

1. Pre-culture transformed cells in 3ml LB medium at 37°C for 18h.
2. 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).
3. Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
4. Irradiate cells on the agar with UV light at desired energy dosage.
5. Wrap plates in aluminium foil and incubate at 37°C.
6. After 16h, count number of colonies formed on control (not UV-irradiated) and UV-irradiated plates.

放射線耐性菌D.radioduranceのもつDNA-repair proteinであるPprI,PprA,PprM,RecAをそれぞれ導入した大腸菌にUV照射

SOS Promoter assay

8mlのLBでincubate　12h。OD600測定しシャーレ（φ50mm）に移してUV照射。2h振盪培養してOD600測定。 遠心沈殿で上澄を捨て、水1mlを加えてペレットを洗う。アセトン500μl加えて　55℃　vortex　15min　　 アセトン100%でブランク　　 A474測定