Team:Chiba/protocol/phenotype/timedelay/j

From 2008.igem.org

(Difference between revisions)
(→プロトコル Protocol [https://2008.igem.org/wiki/index.php?title=Team:Chiba/protocol/phenotype/timedelay/j&action=edit&section=4])
(→翌日,Following day)
Line 30: Line 30:
====翌日,Following day====
====翌日,Following day====
-
 
+
日本語
*T9002
*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。
#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。
Line 38: Line 38:
#48　deep well plateに、1mlずつ分注。
#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).
-
*others
+
日本語
-
#培養液12.5μLを、5mlのLB-Amp液体培地に加え、37℃で6-8時間しんとう培養する。
+
*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。
#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。
Line 47: Line 58:
#48　deep well plateに、所定量分注。
#48　deep well plateに、所定量分注。
-
*測定
+
*cultures containing plasmids you will testing
-
#96 shallow well plateに100μLずつ分注し、蛍光強度を測定する。
+
#Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium.
-
#37°Cでしんとう培養
+
#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。
#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>
*測定条件<Br>
Line 57: Line 82:
::Beam width:Normal Beam
::Beam width:Normal Beam
::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
 +
**pre-shaking:On time = 1min,Off time = 10 sec,
 +
**integration time = 1000ms
 +
**Beam width:Normal Beam
 +
**Wavelength pair = 485nm(excitation) and 527nm(emission)
=== issues? discussion ===
=== issues? discussion ===
蛍光強度がいくつ以上なら目視で確認できるのか-->閾値
蛍光強度がいくつ以上なら目視で確認できるのか-->閾値

Revision as of 05:43, 26 October 2008

>team chiba Internal
>return to 実験ログ
>return to protocols page

Contents

Time-delay check

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)
    • 46-well plate(deep well)
    • Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
  • 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 )。
  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
    1. Aliquote 10mL of the culture into 50mL four 50mL falcon tubes.
    2. Cultures are centrifuged at 3500rpm for 6 minutes.
    3. Dinspense supernatant.
    4. Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
  4. 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。
  • 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
    1. Cultures are centrifuged at 3500rpm for 6 minutes.
    2. Dinspense supernatant.
    3. Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
  4. repeat washing process twice.
  5. Cultures are centrifuged at 3500rpm for 6 minutes.
  6. Dinspense supernatant.
  7. Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
  8. 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
    • pre-shaking:On time = 1min,Off time = 10 sec,
    • integration time = 1000ms
    • Beam width:Normal Beam
    • Wavelength pair = 485nm(excitation) and 527nm(emission)

issues? discussion

蛍光強度がいくつ以上なら目視で確認できるのか-->閾値

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