Team:Osaka/Protocols

From 2011.igem.org

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== Cell survival assay ==
== Cell survival assay ==
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#Pre-culture transformed cells in 3ml LB medium at 37&deg;C for 18h.
+
#Pre-culture transformed cells in 3ml of LB medium at 37&deg;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).
#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.
#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.
#Irradiate cells on the agar with UV light at desired energy dosage.
#Wrap plates in aluminium foil and incubate at 37&deg;C.
#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.
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#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.
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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 ==
== SOS Promoter assay ==
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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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#Pre-culture transformed cells in 8ml of LB medium at 37&deg;C for 12h.
 +
#Transfer pre-culture to OD600 measurement dish (&oslash;50mm)
 +
#Irradiate with UV light at desired energy dosage.
 +
#Incubate irradiated cells for a further 2h.
 +
#Measure OD600 as a measure of cell density.
 +
#Transfer cells into 15ml Falcon tubes and centrifuge.
 +
#Discard supernatant, add 1ml water to wash cells.
 +
#Repeat centrifugation and decantation.
 +
#Add 500μl acetone and vortex.
 +
#Incubate at 55&deg;C for 15min.
 +
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 measurements.
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Revision as of 15:56, 5 October 2011

Cell survival assay

  1. Pre-culture transformed cells in 3ml of 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 (non-irradiated) and UV-irradiated plates.


SOS Promoter assay

  1. Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
  2. Transfer pre-culture to OD600 measurement dish (ø50mm)
  3. Irradiate with UV light at desired energy dosage.
  4. Incubate irradiated cells for a further 2h.
  5. Measure OD600 as a measure of cell density.
  6. Transfer cells into 15ml Falcon tubes and centrifuge.
  7. Discard supernatant, add 1ml water to wash cells.
  8. Repeat centrifugation and decantation.
  9. Add 500μl acetone and vortex.
  10. Incubate at 55°C for 15min.
  11. Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 measurements.
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