Team:HokkaidoU Japan/Notebook/asB0034/asB0034,32-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>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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asB0034_F Tm: 68.7&#8451; & asB0034_R Tm: 68.7&#8451; asB0032_F Tm: 71.6&#8451; & asB0032_R Tm: 70.8&#8451;
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asB0034_F Tm: 68.7&#8451; & asB0034_R Tm: 68.7&#8451;  
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</div>
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<div class="step-recipe">
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asB0032_F Tm: 71.6&#8451; & asB0032_R Tm: 70.8&#8451;
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Ligate pSB1A3 withR0010-stem-asB0034-stem-B0015  Ligate pSB1A3 withR0010-stem-asB0032-stem-B0015
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Ligate pSB1A3 with R0010-stem-asB0034-stem-B0015   
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<div class="step-recipe">
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Ligate pSB1A3 with R0010-stem-asB0032-stem-B0015
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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ligation products 5.0&micro;L to DH5&alpha;
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ligation products  
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5.0&micro;L to DH5&alpha;
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      </ul>
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R0010-stem-asB0034-stem-B0015(on pSB1A3) 100up_F Tm:62.8&#8451; & asB0034_R Tm:68.7&#8451; R0010-stem-asB0032-stem-B0015(on pSB1A3) 100up_F Tm:74.5&#8451; & asB0032_R Tm:70.8&#8451; Kapa-Taq
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R0010-stem-asB0034-stem-B0015(on pSB1A3) 100up_F Tm:62.8&#8451; & asB0034_R Tm:68.7&#8451;
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<div class="step-recipe">
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R0010-stem-asB0032-stem-B0015(on pSB1A3) 100up_F Tm:74.5&#8451; & asB0032_R Tm:70.8&#8451;
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<div class="step-recipe">
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Kapa-Taq
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      </ul>
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R0010-stem-asB0034-stem-B0015(on pSB1A3) R0010-stem-asB0032-stem-B0015(on pSB1A3)
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R0010-stem-asB0034-stem-B0015(on pSB1A3)  
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<div class="step-recipe">
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R0010-stem-asB0032-stem-B0015(on pSB1A3)
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      </ul>
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R0010-stem-asB0034-stem-B0015(on pSB1A3) R0010-stem-asB0032-stem-B0015(on pSB1A3)
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R0010-stem-asB0034-stem-B0015(on pSB1A3)  
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</div>
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<div class="step-recipe">
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R0010-stem-asB0032-stem-B0015(on pSB1A3)
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Latest revision as of 15:29, 9 September 2015

Notebook
Lab Documents

Anti-Sense B0034 (B0032) Construction

  • Start

  • Primer Annealing

    asB0034_F Tm: 68.7℃ & asB0034_R Tm: 68.7℃
    asB0032_F Tm: 71.6℃ & asB0032_R Tm: 70.8℃

  • Digestion

    Cut vector with NcoI, XhoI (using 10×cut smart buffer)

  • Gel extraction

    digested pSB1A3

  • Ethanol Precipitation

    digested pSB1A3

  • Ligation

    Ligate pSB1A3 with R0010-stem-asB0034-stem-B0015
    Ligate pSB1A3 with R0010-stem-asB0032-stem-B0015

  • Transformation

    ligation products
    5.0µL to DH5α

  • Colony PCR

    R0010-stem-asB0034-stem-B0015(on pSB1A3) 100up_F Tm:62.8℃ & asB0034_R Tm:68.7℃
    R0010-stem-asB0032-stem-B0015(on pSB1A3) 100up_F Tm:74.5℃ & asB0032_R Tm:70.8℃
    Kapa-Taq

  • Liquid Culture

    R0010-stem-asB0034-stem-B0015(on pSB1A3)
    R0010-stem-asB0032-stem-B0015(on pSB1A3)

  • Mini-Prep

    R0010-stem-asB0034-stem-B0015(on pSB1A3)
    R0010-stem-asB0032-stem-B0015(on pSB1A3)

  • Complete!

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