Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

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{{Team:HokkaidoU_Japan/CSS}}
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{{Team:HokkaidoU_Japan/Top_Header/CSS}}
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<html>
<html>
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<link href='http://fonts.googleapis.com/css?family=Merriweather' rel='stylesheet' type='text/css'>
<div id="background">
<div id="background">
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<div id="header-top" style="background-color:#6CC4C1;">
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<div id="header-top">
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<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_Header_Top.png">
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</div>
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<div id="header-bottom">
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  <div id="header-bottom-contents">
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    <div class="header-title">
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      <div class="header-title-top">
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      Notebook
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      </div>
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      <div class="header-title-bottom">
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      Lab Documents
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      </div>
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    </div>
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    <div class="header-picture">
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      <img src="https://static.igem.org/mediawiki/2014/4/44/HokkaidoU_Header_ki.png">
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                                 <li>
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                                 <span class="menu-list"><a href="https://2014.igem.org/Team:HokkaidoU_Japan"><span style="position:relative; top:10px;">Top</span></a></span><!-- Increases to 510px in width-->
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                                 <span class="menu-list" ><a href="https://2014.igem.org/Team:HokkaidoU_Japan"><span style="position:relative; top:10px;" >Top</span></a></span><!-- Increases to 510px in width-->
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<ul style="margin-left:20px;">
<ul style="margin-left:20px;">
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Overview</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Introduction</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
</ul>
</ul>
<ul>
<ul>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-Stem System</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
                                                         <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Project/H_Stem#Modeling">Modelling</a></li>
 
</ul>
</ul>
<ul>
<ul>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Anti-senseB0034</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Overview">Overview</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Method">Method</a></li>
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Results">Results</a></li>
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Conclusion">Conclusion</a></li>  
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li>
-
                                                </ul>
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</ul>
<ul>
<ul>
-
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Overview">Length Variation</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
-
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Overview">Overview</a></li>
+
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Overview</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Method">Method</a></li>
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Results">Results</a></li>
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li>
-
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Conclusiton">Conclusion</a></li>
+
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li>
</ul>
</ul>
<ul>
<ul>
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<li class="menu_button">
<li class="menu_button">
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<span class="menu-list">Policy and Practice</span>
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span>
<div class="ldd_submenu" style="width:590px; left:-230px;">
<div class="ldd_submenu" style="width:590px; left:-230px;">
<ul style="margin-left:20px;">
<ul style="margin-left:20px;">
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Education">Education</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Education#School_Festival">School Festival</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Education#High_School">High-school</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li>
</ul>
</ul>
<ul>
<ul>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Survey">Survey</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High School</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Survey#School_Festival">School Festival</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Survey#High_School">High-school</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
</ul>
</ul>
<ul>
<ul>
-
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion">Discussion</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion#Background">Background</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion#Estimation">Estimation</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li>
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<div class="wrapper">
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   <div id="hokkaidou-contents">
   <div id="hokkaidou-contents">
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<h2>anti-sense mRFP Construction</h2>
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  <h2>
 +
Anti-Sense mRFP Construction
 +
</h2>
     <div class="workflow-container">
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       <ul class="steps">
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  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Primer Annealing
+
Get anti-sense vector
</h2>
</h2>
  </div>
  </div>
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        <li>
        <li>
  <time>
  <time>
-
Sun Aug 17
+
Fri Jun 20
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
asB0034-f Tm: 68.7&deg;C asB0034-r Tm: 68.7&deg;C
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Great thanks to N. Nakashima
 +
</div>
 +
    </div>
 +
  </div>
 +
</li>
 +
<li class="step">
 +
  <div class="step-tile">
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  <h2 class="step-title">
 +
PCR & PCR purification
 +
</h2>
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  </div>
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  <div class="step-detail">
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    <div class="step-detail-body">
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      <ul class="step-detail-trials">
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        <li>
 +
  <time>
 +
Fri Aug 15
 +
</time>
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</li>
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      </ul>
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      <div class="step-recipe">
 +
R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;  
 +
</div>
 +
<div class="step-recipe">
 +
KOD plus Neo
 +
</div>
 +
    </div>
 +
  </div>
 +
</li>
 +
<li class="step">
 +
  <div class="step-tile">
 +
  <h2 class="step-title">
 +
Ethanol Precipitation
 +
</h2>
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  </div>
 +
  <div class="step-detail">
 +
    <div class="step-detail-body">
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      <ul class="step-detail-trials">
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        <li>
 +
  <time>
 +
Fri Aug 15
 +
</time>
 +
</li>
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      </ul>
 +
      <div class="step-recipe">
 +
asmRFP
</div>
</div>
    </div>
    </div>
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        <li>
  <time>
  <time>
-
Sun Aug 17
+
Fri Aug 15
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut pHN1257 with NcoI, XhoI(using 10&times;cut smart)
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)
 +
</div>
 +
<div class="step-recipe">
 +
Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
</div>
</div>
    </div>
    </div>
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  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Gel extraction
+
Gel Extraction
</h2>
</h2>
  </div>
  </div>
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        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
digested pHN1257
+
pHN1257 asmRFP
</div>
</div>
    </div>
    </div>
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        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
digested pHN1257
+
asmRFP
</div>
</div>
    </div>
    </div>
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  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Ligation
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Dephosphorylation
</h2>
</h2>
  </div>
  </div>
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        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ligate pHN1257 with asB0034
+
asmRFP
</div>
</div>
    </div>
    </div>
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  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Transformation
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Ligation
</h2>
</h2>
  </div>
  </div>
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        <li>
        <li>
  <time>
  <time>
-
Tue Aug 19
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0&micro;L DNA to DH5&alpha; 42&deg;C        1min on ice 2min
+
Ligate asmRFP with pHN1257
</div>
</div>
    </div>
    </div>
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  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Colony PCR
+
Transformation
</h2>
</h2>
  </div>
  </div>
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        <li>
  <time>
  <time>
-
Wed Aug 20
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7
+
asmRFP(on pHN1257)  
 +
</div>
 +
<div class="step-recipe">
 +
5&micro;L to DH5&alpha; Turbo
</div>
</div>
    </div>
    </div>
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  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Culture
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Colony PCR
</h2>
</h2>
  </div>
  </div>
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  <time>
  <time>
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Thu Aug 21
+
Sun Aug 17, Mon Aug 18
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem
+
asmRFP(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & pHN1257_terminator_R Tm:71.4&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
Kapa-Taq
</div>
</div>
    </div>
    </div>
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  <h2 class="step-title">
  <h2 class="step-title">
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mini-Prep
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Liquid Culture
</h2>
</h2>
  </div>
  </div>
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{{Team:HokkaidoU_Japan/JS}}
{{Team:HokkaidoU_Japan/JS}}

Latest revision as of 15:23, 9 September 2015

Notebook
Lab Documents

Anti-Sense mRFP Construction

  • Start

  • Get anti-sense vector

    Great thanks to N. Nakashima

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    KOD plus Neo

  • Ethanol Precipitation

    asmRFP

  • Digestion

    Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart)
    Cut asmRFP with NcoI, XhoI (using 10×Cut Smart)

  • Gel Extraction

    pHN1257 asmRFP

  • Ethanol Precipitation

    asmRFP

  • Dephosphorylation

    asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) pHN1257_up_F Tm:60.1℃ & pHN1257_terminator_R Tm:71.4℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-Prep

    asmRFP(pHN1257)

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/asmRFP"