Team:Chiba/jk/γ/Trl

From 2008.igem.org

(Difference between revisions)
(Protocol)
(装置&試薬(apparatus&reagents))
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===装置&試薬(apparatus&reagents)===
===装置&試薬(apparatus&reagents)===
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'''装置(apparatus)'''
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*Equipment
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*しんとう培養器(shaking incubator)(37℃,30℃)  
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:shaking incubator
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*48 well plate(deep well)  
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::Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
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*Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)  
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:46-well plate(deep well)
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:96-well plate(deep well)  
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:Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)  
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:Beckman Allegratm X-12R Centrifuga(Beckman Coulter)
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'''試薬(reagents)'''  
'''試薬(reagents)'''  
*AHL(100uM,5uM,100nM)  
*AHL(100uM,5uM,100nM)  

Revision as of 23:52, 29 October 2008

実験ログ
出力班


Contents

Time Responce Liquid

purpose

インダクションをかけてからいち早く、確認できる出力を見つけること
To find the earliest output gene after induction

装置&試薬(apparatus&reagents)

  • Equipment
shaking incubator
Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
46-well plate(deep well)
96-well plate(deep well)
Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
Beckman Allegratm X-12R Centrifuga(Beckman Coulter)


試薬(reagents)

  • AHL(100uM,5uM,100nM)
  • IPTG(100nM)
  • X-gal
  • M9
  • PBS

Method

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G KanR

  • Liquid medium experiment
  1. Pre-culture
    1. Picked and cultured the following plate in 2mL of LB:
      1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for 12h.
  2. Culture
    1. Dilute pre-cultures and add to new LB medium.
      1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for about 6 h
  3. Wash
    1. Transfer 10mL each of the culture to 50mL centrifuge tubes.
    2. Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
    3. Add physiological saline and resuspention
    4. Repeated wash twice.
    5. Add M9 minimal medium.
  4. Mix
    1. Dispens each culture into 48-well deep well or 96-well deep well
    2. Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
    3. Add AHL fainal conc. 100nM ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
  5. Measure fluorescence intensity every 1 h.






Result

ホーム メンバー紹介 プロジェクト紹介 Parts Submitted to the Registry モデリング ノート