Team:Chiba/protocol/phenotype/timedelay/j
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- | == | + | <html><link rel="stylesheet" href="https://2008.igem.org/wiki/index.php?title=User:Maiko/chiba.css&action=raw&ctype=text/css" type="text/css" /></html> |
+ | >[[Team:Chiba/protocol/j|プロトコルページへ]]<Br> | ||
- | === | + | == 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 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 === | |
- | 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。 | + | ====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。 | :-->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。 | |
- | + | ||
- | *培養液12.5μLを、5mlのLB- | + | 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。 | :-->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 | -->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<Br> | ||
::測定前-->shake:On time = 1min,Off time = 10 sec, | ::測定前-->shake:On time = 1min,Off time = 10 sec, | ||
::測定-->integration time = 1000ms | ::測定-->integration time = 1000ms | ||
Line 48: | Line 86: | ||
::Wavelength pair = 485nm(excitation) and 527nm(emission) | ::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) |
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)