Team:HokkaidoU Japan/Notebook/H Stem/Construction

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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li>
<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/Outreach/Discussion#Estimation">Evaluation</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>
</ul>
</ul>
</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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mini-Prep
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Mini-Prep
</h2>
</h2>
  </div>
  </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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1C3 Vector
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pSB1C3 vector
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac (plasmid) with 100up_F & 200dn_R, Ptet-B0034-mRFP-dT (plasmid) with XhoI-RBS & NcoI-mRFP (anti-sense), double-Terminator (plasmid) with 100up & 200dn
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R0010 (plasmid) with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
        R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0&#8451; & XhoI_RBS_R Tm:68.2&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
        B0015 (plasmid) with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac with SpeI, anti-sense with NcoI, anti-sense with XhoI, dT with XbaI
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R0010 with SpeI
 +
</div>
 +
<div class="step-recipe">
 +
        anti-sense with NcoI, XhoI
 +
</div>
 +
<div class="step-recipe">
 +
        B0015 with XbaI
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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stem_upstream_F & stem_upstream_R, stem_downstream_F & stem_downstream_R
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stem_upstream_F Tm:71.4&#8451; & stem_upstream_R Tm:71.4&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
stem_downstream_F Tm:71.4&#8451; & stem_downstream_R Tm:71.4&#8451;
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Plac & stem-up & as-up, as-dn & stem-dn & dT
+
R0010 & stem-up & as-up  
 +
</div>
 +
<div class="step-recipe">
 +
as-dn & stem-dn & B0015
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Plac-stem-as with 100up_F & XhoI_RBS_R, as-stem-dT with NcoI_mRFP_F & 200dn_R
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R0010-stem-as with 100up_F Tm:62.6&#8451; & XhoI_RBS_R Tm:68.2&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
as-stem-B0015 with NcoI-mRFP_F Tm:76.0&#8451; & 200dn Tm:66.6&#8451;
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Plac-stem-as & as-stem-dT with 100up_F & 200dn_R
+
R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac-stem-as-stem-dT with EcoRI & PstI, 1C3 with EcoRI & PstI
+
Cut R0010-stem-as-stem-B0015 with EcoRI & PstI
 +
</div>
 +
<div class="step-recipe">
 +
Cut pSB1C3 with EcoRI & PstI
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac-stem-as-stem-dT & 1C3
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Ligate R0010-stem-as-stem-B0015 with pSB1C3
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac-stem-as-stem-dT on 1C3
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R0010-stem-as-stem-B0015(on pSB1C3)
 +
</div>
 +
<div class="step-recipe">
 +
5.0&micro;L to DH5alpha;
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Plac-stem-as-stem-dT on 1C3 with stem_up_F & stem_d_R / 100up_F & 200dn_R / EX_F & PS_R
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R0010-stem-as-stem-B0015(on pSB1C3) with  
 +
</div>
 +
<div class="step-recipe">
 +
stem_up_F Tm:71.4&#8451; & stem_dn_R Tm:71.4&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
EX_F Tm:70.4&#8451; & PS_R Tm:74.6&#8451;
</div>
</div>
    </div>
    </div>
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   <div id="footer-content">
<div id="footer-logo">
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<a href="http://igemhokkaidou.wordpress.com"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a>
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Latest revision as of 15:28, 9 September 2015

Notebook
Lab Documents

H-Stem Construction

  • Start

  • Mini-Prep

    pSB1C3 vector

  • PCR

    R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃
    B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    R0010 with SpeI
    anti-sense with NcoI, XhoI
    B0015 with XbaI

  • Primer Annealing

    stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃
    stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃

  • Ligation

    R0010 & stem-up & as-up
    as-dn & stem-dn & B0015

  • PCR

    R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃
    as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃

  • PCR

    R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    Cut R0010-stem-as-stem-B0015 with EcoRI & PstI
    Cut pSB1C3 with EcoRI & PstI

  • Ligation

    Ligate R0010-stem-as-stem-B0015 with pSB1C3

  • Transformation

    R0010-stem-as-stem-B0015(on pSB1C3)
    5.0µL to DH5alpha;

  • Colony PCR

    R0010-stem-as-stem-B0015(on pSB1C3) with
    stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃
    100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    EX_F Tm:70.4℃ & PS_R Tm:74.6℃

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/H_Stem/Construction"