Team:Chiba/protocol/phenotype/timedelay/j
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- | + | >[[Team:Chiba/protocol/j|プロトコルページへ]]<Br> | |
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== Time-delay check(冨永)== | == Time-delay check(冨永)== | ||
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=== 装置 and 試薬 Equipments and Materials === | === 装置 and 試薬 Equipments and Materials === | ||
- | + | ====装置 Equipment==== | |
- | + | *shaking incubator(37°C,30°C) | |
- | **46-well plate(deep well) | + | **Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C) |
- | + | **タイテック BioShaker BR-33FM(30°C,200rpm) | |
+ | *46-well plate(deep well) | ||
+ | *Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6) | ||
+ | *Beckman Allegra<sup>tm</sup> X-12R Centrifuga(Beckman Coulter) | ||
- | + | ====試薬 Materials==== | |
- | + | *AHL(100uM) | |
- | + | *E.coli Culture Containing T9002 | |
- | + | *E.coli Culture Containing plasmids you will testing | |
=== プロトコル Protocol === | === プロトコル Protocol === | ||
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#48 deep well plateに、所定量分注。 | #48 deep well plateに、所定量分注。 | ||
+ | |||
+ | English | ||
*cultures containing plasmids you will testing | *cultures containing plasmids you will testing | ||
#Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium. | #Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium. | ||
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#Add 5mL of new LB-ampicillin medium and resuspense with pipetting. | #Add 5mL of new LB-ampicillin medium and resuspense with pipetting. | ||
#aliquote cultures into a 48-deep well plate(deep well). | #aliquote cultures into a 48-deep well plate(deep well). | ||
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====測定,Measurement==== | ====測定,Measurement==== | ||
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#Measure fluorescence intensity. | #Measure fluorescence intensity. | ||
*conditions | *conditions | ||
- | ** | + | **shaking(before measurement):On time = 1min,Off time = 10 sec, |
**integration time = 1000ms | **integration time = 1000ms | ||
**Beam width:Normal Beam | **Beam width:Normal Beam | ||
**Wavelength pair = 485nm(excitation) and 527nm(emission) | **Wavelength pair = 485nm(excitation) and 527nm(emission) | ||
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Latest revision as of 09:28, 15 December 2008
Contents |
Time-delay check(冨永)
目的 Purpose
To Confirm that communication using non-endogenous molecules results in slower activation of receivers.
装置 and 試薬 Equipments and Materials
装置 Equipment
- shaking incubator(37°C,30°C)
- Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
- タイテック BioShaker BR-33FM(30°C,200rpm)
- 46-well plate(deep well)
- Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
- Beckman Allegratm X-12R Centrifuga(Beckman Coulter)
試薬 Materials
- AHL(100uM)
- E.coli Culture Containing T9002
- E.coli Culture Containing plasmids you will testing
プロトコル Protocol
プレ培(O/N,測定日の前日),Culturing overnight (before the testing day):
- グリセロールストックからpickして、37℃しんとう培養器で一晩培養する(LB-Amp液体培地,2mL,37°C )。
- Inoculate all cultures you will testing from gloycerol stocks into 2mL of LB-ampicillin liquid medium.
- Also inoculate a culture containing T9002 into 2mL of LB-ampicillin liquid medium.
- Incubate all cultures with shaking at 37°C(O/N).
翌日,Following day
日本語
- T9002
- 培養液100μLを、40mlのLB-Amp液体培地に加え、37℃で6-8時間しんとう培養する。
- Wash(T9002)-->培養液を50mlファルコンチューブに、10mlずつ分注。
- -->3500rpmで6分間遠心。上澄みを捨てる。
- 新しいLB-Amp培地を10mlずつ加え(1倍希釈)、pipettingにより再懸濁。
- 48 deep well plateに、1mlずつ分注。
English:
- cultures containing T9002
- Inoculate a culture by adding 100μL of the cultures into 40mL of LB-ampicillin medium.(in a flask)
- Incubate a culture for 6-8 hours with shaking at 37°C.
- Wash
- Aliquote 10mL of the culture into 50mL four 50mL falcon tubes.
- Cultures are centrifuged at 3500rpm for 6 minutes.
- Dinspense supernatant.
- Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
- aliquote 1mL of the cultures into a 48-deep well plate(deep well).
日本語
- cultures containing plasmids you will testing
- 培養液12.5μLを、5mlのLB-Amp液体培地に加え、37°Cで6-8時間しんとう培養する。
- Wash-->3500rpmで6分間遠心。上澄みを捨てる。
- -->新しいLB-Amp培地を3mlずつ加え、pipettingにより再懸濁。
-->この操作を二回繰り返す
- 新しいLB-Amp培地を10mlずつ加え(1倍希釈)、pipettingにより再懸濁。
- 48 deep well plateに、所定量分注。
English
- cultures containing plasmids you will testing
- Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium.
- Incubate cultures for 6-8 hours with shaking at 37°C.
- Wash
- Cultures are centrifuged at 3500rpm for 6 minutes.
- Dinspense supernatant.
- Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
- repeat washing process twice.
- Cultures are centrifuged at 3500rpm for 6 minutes.
- Dinspense supernatant.
- Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
- aliquote cultures into a 48-deep well plate(deep well).
測定,Measurement
日本語:
- 37°Cでしんとう培養
- 96 shallow well plateに100μLずつ分注し、蛍光強度を測定する。
- 2h後、3h後…に、100μLずつ分注し、蛍光強度を測定する。
- 測定条件
- 測定前-->shake:On time = 1min,Off time = 10 sec,
- 測定-->integration time = 1000ms
- Beam width:Normal Beam
- Wavelength pair = 485nm(excitation) and 527nm(emission)
English:
- Incubate testing cultures with shaking at 37°C.
- After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
- Measure fluorescence intensity.
- conditions
- shaking(before measurement):On time = 1min,Off time = 10 sec,
- integration time = 1000ms
- Beam width:Normal Beam
- Wavelength pair = 485nm(excitation) and 527nm(emission)