Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment

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{{Team:HokkaidoU_Japan/CSS}}
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{{Team:HokkaidoU_Japan/Top_Header/CSS}}
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{{Team:HokkaidoU_Japan/LargeDropDown_Projects/CSS}}
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{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}
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      Notebook
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      Lab Documents
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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>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li>
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                                                        <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>
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<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>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Method">Method</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Results">Results</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034/Conclusion">Conclusion</a></li>  
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                                                             <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>
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<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>
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            <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>
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                                                             <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>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li>
</ul>
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<ul>
<ul>
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<span class="menu-list">Policy and Practice</span>
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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>
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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>
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<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_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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      </ul>
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      <div class="step-recipe">
      <div class="step-recipe">
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R0010-B0034-E1010-B0015(pSB6A1) from destribution kit 1&micro;L DNA to JM109
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R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1&micro;L to JM109
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
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pSB6A1
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R0010-B0034-E1010-B0015(on pSB6A1)
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      </ul>
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      <div class="step-recipe">
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pSB6A1
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R0010-B0034-E1010-B0015(on pSB6A1)
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      </ul>
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      <div class="step-recipe">
      <div class="step-recipe">
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R0040-B0034-E1010-B0015 as RBS XhoI, as mRFP NcoI
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R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76/0&#8451;     
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</div>
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<div class="step-recipe">
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KOD plus Neo
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      </ul>
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      <div class="step-recipe">
      <div class="step-recipe">
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anti-sense(mRFP)
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asmRFP
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart) Cut anti-sense(mRFP) with NcoI, XhoI (using 10&times;Cut Smart)
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)  
 +
</div>
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<div class="step-recipe">
 +
Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
</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 & Ethanol precipetation
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Gel Extraction
</h2>
</h2>
  </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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pHN1257 anti-sense(mRFP)
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pHN1257 & asmRFP
</div>
</div>
    </div>
    </div>
  </div>
  </div>
</li>
</li>
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<li class="step">
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  <div class="step-tile">
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  <h2 class="step-title">
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Ethanol precipetation
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</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>
 +
Sun Jul 27
 +
</time>
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</li>
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      </ul>
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      <div class="step-recipe">
 +
pHN1257 & asmRFP
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</div>
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    </div>
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  </div>
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</li>
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<li class="step">
<li class="step">
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Ligate anti-sense(mFP) with pHN1257
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Ligate asmRFP with pHN1257
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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anti-sense(mRFP) on pHN1257 5&micro;L DNA to DH5&alpha; Turbo
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asmRFP(on pHN1257)
 +
</div>
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<div class="step-recipe">
 +
5&micro;L to DH5&alpha; Turbo
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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anti-sense(mRFP) on pHN1257
+
asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;
 +
</div>
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<div class="step-recipe">
 +
Kapa-Taq
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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anti-sense(mRFP) on pHN1257
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asmRFP(on pHN1257)
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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anti-sense(mRFP) on pHN1257
+
asmRFP(on pHN1257)
</div>
</div>
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  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
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Transformation
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DoubleTransformation
</h2>
</h2>
  </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257 & pSB6A1 2.5&micro;L each DNA to DH5&alpha; Turbo
+
asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
 +
</div>
 +
<div class="step-recipe">
 +
2.5&micro;L each to DH5&alpha; Turbo
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Culture anti-sense(mRFP) on pHN1257 & pSB6A1 1. 2mL LB with 100&micro;L 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.
+
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
 +
</div>
 +
<div class="step-recipe">
 +
1. 2mL LB with 100&micro;L 100mM IPTG  
 +
2. 2mL LB However, IPTG was too much to grow normally.
 +
 
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Culture anti-sense(mRFP) on pHN1257 & pSB6A1 E. coli was early growth 1. 2mL LB with 20&micro;L 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.
+
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20&micro;L 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.
 +
</div>
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</div>
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Latest revision as of 15:25, 9 September 2015

Notebook
Lab Documents

Wrong Plac failed Experiment

  • Start

  • Get anti-sense vector

    pHN1257 Great thanks to N. Nakashima

  • Transformation

    R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1µL to JM109

  • Liquid Culture

    R0010-B0034-E1010-B0015(on pSB6A1)

  • Mini-prep

    R0010-B0034-E1010-B0015(on pSB6A1)

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃
    KOD plus Neo

  • Ehanol precipetation

    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 precipetation

    pHN1257 & asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-prep

    asmRFP(on pHN1257)

  • DoubleTransformation

    asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    2.5µL each to DH5α Turbo

  • Asssay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.

  • Assay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20µL 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.

  • Failure...

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/Plac-failed-experiment"