Team:Osaka/Protocols

(Difference between revisions)

Revision as of 15:44, 5 October 2011

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

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

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 == Cell survival assay ==
-まくパーツをLB3mlでプレ培養18h。照射するエネルギー量に応じて希釈濃度を変える。 プレートに50μｌまいて乾燥させてからＵＶ照射。 可視光を当てないようにアルミ箔で包んでincubate　37℃
+#Pre-culture transformed cells in 3ml LB medium at 37&deg;C for 18h.
-As an initial screen for DNA repair protein activity , we used the UV assay. The cells were plated on respective agar plates at different dilutions, air dried, and then exposed to different doses of UV radiation. Plates were wrapped with aluminum foil and incubated in the dark. Colonyforming units were scored after 16h incubation at 37℃.
+#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&deg;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