Team:UT-Tokyo/Protocol

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                     <li><span class="mhead">Project Description</span>
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                     <li><span class="mhead">Protocols</span>
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                         <li><a href="#Background">Background</a></li>
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                         <li><a href="#qRT-PCR">qRT-PCR</a></li>
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                        <li><a href="#Project_Summary">Project Summary</a></li>
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<h1 id="Project_Description">Project Description</h1>
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<h1 id="Protocols">Protocols</h1>
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             <h2 id="Background">Background</h2>
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             <h2 id="qRT-PCR">qRT-PCR</h2>
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             <p class="ini"> Genetic engineering has achieved numerous victories both in basic and applied biology. However, there still remains room for progress in the area, especially in developing new tools. For instance, although the importance of cell-cell interaction has been emphasized, there are limited number of tools in bioengineering to utilize it. Controlling interaction among microorganisms will help understanding living things and constructing completely novel multicellular systems. </p>
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             <p class="ini"> 1. pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin<br>
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2. incubate at 37 degree over night<br>
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3. dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin<br>
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4. grow to OD600=0.6 at 37 degree<br>
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5. centrifuge 1mL culture at 15,000rpm for 1min<br>
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6. remove the supernatant<br>
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7. resuspend with TE buffer containing lysozyme(0.4mg/mL)<br>
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8. incubate at room temperature for 5min<br>
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9. isolate total RNA from the lysate with RNeasy® Mini<br>
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10. synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit<br>
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11. quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix </p>
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            <h2 id="Project_Summary">Project Summary</h2>
 
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            <p class="ini">In our project, we are trying to design cell-cell interaction-based spatiotemporal control of biological reactions. We are now constructing an E. coli cell-based system which functions as an analogue clock with a clock hand moving clockwise. We anticipate that this will be achieved by combining positive and negative feedback loops and quorum sensing-based cell-cell communication.''</p>
 
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Revision as of 08:03, 13 October 2013

           PROTOCOL

Protocols

qRT-PCR

1. pick up a colony and inoculate in LB broth containing 100µg/mL ampicillin
2. incubate at 37 degree over night
3. dillute the over night culture (1:50) in LB broth containing 100µg/mL ampicillin
4. grow to OD600=0.6 at 37 degree
5. centrifuge 1mL culture at 15,000rpm for 1min
6. remove the supernatant
7. resuspend with TE buffer containing lysozyme(0.4mg/mL)
8. incubate at room temperature for 5min
9. isolate total RNA from the lysate with RNeasy® Mini
10. synthesize cDNA from 1µg total RNA with PrimeScript® 1st strand cDNA Synthesis Kit
11. quantify the cDNA of the target gene or 16S rRNA with Power SYBR® Green PCR Master Mix

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