Team:Osaka/Protocols
From 2011.igem.org
(Difference between revisions)
Line 2: | Line 2: | ||
<div class="padding"> | <div class="padding"> | ||
== Cell survival assay == | == Cell survival assay == | ||
- | + | #Pre-culture transformed cells in 3ml LB medium at 37°C for 18h. | |
- | + | #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). | |
+ | #Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. | ||
+ | #Irradiate cells on the agar with UV light at desired energy dosage. | ||
+ | #Wrap plates in aluminium foil and incubate at 37°C. | ||
+ | #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 | 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 |
Revision as of 15:44, 5 October 2011
Cell survival assay
- Pre-culture transformed cells in 3ml LB medium at 37°C for 18h.
- 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).
- Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
- Irradiate cells on the agar with UV light at desired energy dosage.
- Wrap plates in aluminium foil and incubate at 37°C.
- 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