Team:Osaka/Protocols

From 2011.igem.org

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(Cell survival assay)
 
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== Protocols ==
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=== Cell survival assay 1: UV irradiation ===
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#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.
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#Induce parts with IPTG addition (to final concentration of 100&micro;M) for 1h.
 +
#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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#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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#Irradiate cells on the agar with UV light at desired energy dosage.
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#Wrap plates in aluminium foil and incubate at 37&deg;C.
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#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.
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== Cell survival assay ==
 
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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℃
 
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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℃.
 
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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
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=== Cell survival assay 2: Mitomycin C ===
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#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.
 +
#Induce parts with IPTG addition to final concentration of 100&micro;M and incubate for 1h.
 +
#Add mitomycin C to desired final concentration (we used 2&micro;g/ml) and incubate at 37&deg;C for a further 2h.
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#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
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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).
 +
#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&deg;C.
 +
#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.
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== SOS Promoter assay ==
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3mlのLBでプレ培養。それを40mlのLBに100μl加えてincubate 20h。OD600測定し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vortex。もう一度遠心沈殿で上澄捨てる。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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=== SOS promoter assay 1: Carotenoid biosynthesis ===
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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 surrogate for 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.
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#Incubate at 55&deg;C for 15min.
 +
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 values.
 +
 
 +
 
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=== SOS promoter assay 2: GFP ===
 +
#Pre-culture transformed cells in 20ml 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 surrogate for cell density.
 +
#Transfer cells into 50ml Falcon tubes and centrifuge.
 +
#Discard supernatant, add 4ml water to wash cells.
 +
#Repeat centrifugation and decantation.
 +
#Resuspend cells in 4ml water.
 +
#Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.
 +
</div>

Latest revision as of 23:45, 28 October 2011

Protocols

Cell survival assay 1: UV irradiation

  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) for 1h.
  3. 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).
  4. Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
  5. Irradiate cells on the agar with UV light at desired energy dosage.
  6. Wrap plates in aluminium foil and incubate at 37°C.
  7. After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.


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.


SOS promoter assay 1: Carotenoid biosynthesis

  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 surrogate for 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 values.


SOS promoter assay 2: GFP

  1. Pre-culture transformed cells in 20ml 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 surrogate for cell density.
  6. Transfer cells into 50ml Falcon tubes and centrifuge.
  7. Discard supernatant, add 4ml water to wash cells.
  8. Repeat centrifugation and decantation.
  9. Resuspend cells in 4ml water.
  10. Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.