Team:Chiba/protocol/phenotype/timedelay/j

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目的 Purpose
To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

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 ).
 * 1) Inoculate all cultures you will testing from gloycerol stocks into 2mL of LB-ampicillin liquid medium.
 * 2) Also inoculate a culture containing T9002 into 2mL of LB-ampicillin liquid medium.
 * 3) Incubate all cultures with shaking at 37°C(O/N).

翌日,Following day
日本語
 * T9002
 * 1) 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.
 * 2) 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.


 * 1) 新しいLB-Amp培地を10mlずつ加え（1倍希釈）、pipettingにより再懸濁.
 * 2) 48　deep well plateに、1mlずつ分注.

English:
 * cultures containing T9002
 * 1) Inoculate a culture by adding 100μL of the cultures into 40mL of LB-ampicillin medium.(in a flask)
 * 2) Incubate a culture for 6-8 hours with shaking at 37°C.
 * 3) Wash
 * 4) Aliquote 10mL of the culture into 50mL four 50mL falcon tubes.
 * 5) Cultures are centrifuged at 3500rpm for 6 minutes.
 * 6) Dinspense supernatant.
 * 7) Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
 * 8) aliquote 1mL of the cultures into a 48-deep well plate(deep well).

日本語
 * cultures containing plasmids you will testing
 * 1) 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.
 * 2) 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
 * 1) 新しいLB-Amp培地を10mlずつ加え（1倍希釈）、pipettingにより再懸濁.
 * 2) 48　deep well plateに、所定量分注.

English
 * cultures containing plasmids you will testing
 * 1) Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium.
 * 2) Incubate cultures for 6-8 hours with shaking at 37°C.
 * 3) Wash
 * 4) Cultures are centrifuged at 3500rpm for 6 minutes.
 * 5) Dinspense supernatant.
 * 6) Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
 * 7) repeat washing process twice.
 * 8) Cultures are centrifuged at 3500rpm for 6 minutes.
 * 9) Dinspense supernatant.
 * 10) Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
 * 11) aliquote cultures into a 48-deep well plate(deep well).

測定,Measurement
日本語:
 * 1) 37°Cでしんとう培養
 * 2) 96 shallow well plateに100μLずつ分注し、蛍光強度を測定する.
 * 3) 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:
 * 1) Incubate testing cultures with shaking at 37°C.
 * 2) After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
 * 3) 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)