Team:HokkaidoU Japan/Notebook/Length/as90 120

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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
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<h2>
Anti-sense 90, 120 Construction
Anti-sense 90, 120 Construction
</h2>
</h2>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0040-B0034-E1010-B0015 as RBS XhoI, 90bp as mRFP Nco as RBS XhoI, 120bp as mRFP Nco
+
R0040-B0034-E1010-B0015
 +
</div>
 +
<div class="step-recipe">
 +
as_RBS_XhoI_F Tm:68.2&#8451; & 90bp_asmRFP_Nco_R Tm:79.9&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
as_RBS_XhoI_F Tm:68.2&#8451; & 120bp_asmRFP_Nco_R Tm:66.9&#8451;
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut as 90, as120, H-stem with NcoI, XhoI(using 10&times;Cut Smart)
+
Cut as90(on H-stem) with NcoI,XhoI(using 10&times;Cut Smart)
 +
</div>
 +
<div class="step-recipe">
 +
        Cut as120(on H-stem) 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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Dephophorylation
+
Dephosphorylation
</h2>
</h2>
  </div>
  </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
H-stem
+
Antactic phosphorylate H-stem (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ligate as90, as120 with H-stem
+
Ligate as90, as120 with H-stem(on pSB1A3)
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
as90, as120 on H-stem 5&micro;L DNA to DH5&alpha; Turbo
+
as90, as120(on pSB1A3)
 +
</div>
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<div class="step-recipe">
 +
5&micro;L DNA to DH5&alpha; Turbo
</div>
</div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
as90, as120 on H-stem
+
as90 (on pSB1A3) XhoI_RBS_ Tm:68.2&#8451; & as90_NcoI_ Tm:79.9&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
        as120(on pSB1A3) XhoI_RBS_ Tm:68.2&#8451; & as120_NcoI_ Tm:66.9&#8451;
</div>
</div>
 +
<div class="step-recipe">
 +
        Kapa-Taq
 +
</div>
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    </div>
    </div>
  </div>
  </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
as90, as120 on H-stem
+
R0010-K1524104-B0015(on pSB1A3)
 +
</div>
 +
<div class="step-recipe">
 +
        R0010-K1524105-B0015(on pSB1A3)
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
as90, as120 on H-stem
+
R0010-K1524104-B0015(on pSB1A3)
 +
</div>
 +
<div class="step-recipe">
 +
        R0010-K1524105-B0015(on pSB1A3)
 +
 
</div>
</div>
    </div>
    </div>
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
as90, as120 on H-stem
+
R0010-K1524104-B0015(on pSB1A3)
 +
</div>
 +
<div class="step-recipe">
 +
        R0010-K1524105-B0015(on pSB1A3)
 +
 
</div>
</div>
    </div>
    </div>
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{{Team:HokkaidoU_Japan/JS}}

Latest revision as of 15:32, 9 September 2015

Notebook
Lab Documents

Anti-sense 90, 120 Construction

  • Start

  • PCR & PCR purification

    R0040-B0034-E1010-B0015
    as_RBS_XhoI_F Tm:68.2℃ & 90bp_asmRFP_Nco_R Tm:79.9℃
    as_RBS_XhoI_F Tm:68.2℃ & 120bp_asmRFP_Nco_R Tm:66.9℃

  • Digestion

    Cut as90(on H-stem) with NcoI,XhoI(using 10×Cut Smart)
    Cut as120(on H-stem) with NcoI,XhoI(using 10×Cut Smart)

  • Ethanol precipitation

    as90, as120

  • Gel Extraction

    H-stem

  • Dephosphorylation

    Antactic phosphorylate H-stem (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate as90, as120 with H-stem(on pSB1A3)

  • Transformation

    as90, as120(on pSB1A3)
    5µL DNA to DH5α Turbo

  • Colony PCR

    as90 (on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as90_NcoI_ Tm:79.9℃
    as120(on pSB1A3) XhoI_RBS_ Tm:68.2℃ & as120_NcoI_ Tm:66.9℃
    Kapa-Taq

  • Liquid Culture

    R0010-K1524104-B0015(on pSB1A3)
    R0010-K1524105-B0015(on pSB1A3)

  • Mini-prep

    R0010-K1524104-B0015(on pSB1A3)
    R0010-K1524105-B0015(on pSB1A3)

  • Dye terminator

    R0010-K1524104-B0015(on pSB1A3)
    R0010-K1524105-B0015(on pSB1A3)

  • Complete!

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