Team:Chiba/Calendar-Home/21 October 2008

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朝　receiver(T9002)LB-Amp  プレ培したものをプレートに撒く。コロニーが1000位になるように
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>[[Team:Chiba|Home]] | [[Team:Chiba/Notebook|Notebook]]
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　　sender(Ptet-LuxI)LB-Amp  10ml培養×3（①washなし、②washあり、③1:10-1:20or50-1:100）
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夜　センダーが濁ったら②③はwash×3
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[[Team:Chiba/Calendar-Home/20 October 2008|20 October 2008 <]]|[[Team:Chiba/Calendar-Home/22 October 2008|> 22 October 2008]]
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　　①は菌液10mlにLB-agar10mlを加える。（Ampも）
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　　②はwash後菌液10mlにLB-agarを10ml加える。（Ampも）
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　　③(1:10)はwashした菌液を1mlとLB-Ampを9mlまぜ、そこにLB-agarを10ml加える。
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　　　(1:20)はwashした菌液を0.5mlとLB-Ampを9.5mlまぜ、そこにLB-agarを10ml加える。
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　　　(1:100)はwashした菌液を0.1mlとLB-Ampを9.9mlまぜ、そこにLB-agarを10ml加える。
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　　また、ネガコンようとしてLB10mlと、LB-agar10mlを混ぜてプレートにしたものを用意する。
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==Laboratory work==
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===Team:Demo-Rs===
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①②③、ネガコンをプレートに移して固まるまで待つ。固まったらreceiver(T9002)をプレートからニトロセルロースで写し取りそれぞれのプレートに2枚ずつはり、その時をスタートとして37℃の培養機の中にいれ、30分ごとにUVを当ててGFPが出ているかどうか確認する。
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====Varying bacterial numbers====
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20 October~
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##Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))was plated so as to produce about 1000 colonies.
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<BR>
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#Sender(S03623) pre-incubation
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深夜　　T9002をtransformation
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##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 50mL  entrifuge tubes in 10mL of LB-Amp (37&deg;C,12h)(2 tubes)
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#Sender Wash
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##Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20&deg;C,3600rpm for 6min and discarded supernatant.
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##Added 10mL LB-Amp to each tube.
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##Repeated wash twice.
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#Creating bacterial plates
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##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-Amp-agar(50&deg;C)(10ml)to produce sender containing bacterialplate-1.
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##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100&mu;l)was mixed with LB-Amp(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-Amp-agar(50&deg;C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate-2.
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##LB-Amp pre-cultured Sender solution-2(10&mu;l) and LB-Amp(9.99ml)was mixed to dilute 1000-fold.10ml of this solution and LB-Amp-agar(50&deg;C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) containing bacterial plate-3
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#Lifted with nitrocellulose
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##Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate (1~3) and Sender-absent negative control plate (t=0). Determined the time required for the colonies to fluoresce depending on the bacterial concentration (100 and 1000-fold dilution).
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#Method to detect fluorescence
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##Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

Latest revision as of 00:41, 30 October 2008

>Home | Notebook

20 October 2008 <|> 22 October 2008

Laboratory work

Team:Demo-Rs

Varying bacterial numbers

20 October~

    1. Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))was plated so as to produce about 1000 colonies.
  2. Sender(S03623) pre-incubation
    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 50mL entrifuge tubes in 10mL of LB-Amp (37°C,12h)(2 tubes)
  3. Sender Wash
    1. Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20°C,3600rpm for 6min and discarded supernatant.
    2. Added 10mL LB-Amp to each tube.
    3. Repeated wash twice.
  4. Creating bacterial plates
    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-Amp-agar(50°C)(10ml)to produce sender containing bacterialplate-1.
    2. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100μl)was mixed with LB-Amp(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-Amp-agar(50°C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate-2.
    3. LB-Amp pre-cultured Sender solution-2(10μl) and LB-Amp(9.99ml)was mixed to dilute 1000-fold.10ml of this solution and LB-Amp-agar(50°C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) containing bacterial plate-3
  5. Lifted with nitrocellulose
    1. Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate (1~3) and Sender-absent negative control plate (t=0). Determined the time required for the colonies to fluoresce depending on the bacterial concentration (100 and 1000-fold dilution).
  6. Method to detect fluorescence
    1. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
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