Team:HokkaidoU Japan/Notebook/asB0034/asE1010 Target gene vector Exchange to 6A1

From 2014.igem.org

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R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5&#8451; & Ptet_SpeI_R Tm:72.6&#8451; (insert) KOD plus Neo
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R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5&#8451; & Ptet_SpeI_R Tm:72.6&#8451; (insert)
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KOD plus Neo
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Cut  vector with EcoRI, PstI (using 10&times;H buffer)  Cut  insert with EcoRI, PstI (using 10&times;M buffer)
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Cut  vector with EcoRI, PstI (using 10&times;H buffer)   
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Cut  insert with EcoRI, PstI (using 10&times;M buffer)
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Mon Oct 6
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Mon Oct 6,Thu Oct 7
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ligation products each of 5.0&micro;L to DH5&alpha;
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ligation products  
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each of 5.0&micro;L to DH5&alpha;
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Tue Oct 7
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ligation products 5.0&micro;L to DH5&alpha;
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Transformation
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Tue Oct 7
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ligation products 5.0&micro;L to DH5&alpha;
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      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
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Cut  vector with EcoRI, PstI (using 10&times;H buffer) Cut  insert with EcoRI, PstI (using 10&times;M buffer)
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Cut  vector with EcoRI, PstI (using 10&times;H buffer)  
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</div>
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<div class="step-recipe">
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Cut  insert with EcoRI, PstI (using 10&times;M buffer)
</div>
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ligation products 5.0&micro;L DNA to DH5&alpha;
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ligation products  
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</div>
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<div class="step-recipe">
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5.0&micro;L DNA to DH5&alpha;
</div>
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R0040-B0032-E1010-B0015(on pSB4C5) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451; R0040-B0034-E1010-B0015(on pSB4C5) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451; R0040-B0032-E1010-B0015(on pSB2K3) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451; R0040-B0034-E1010-B0015(on pSB2K3) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451; Kapa-Taq
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R0040-B0032-E1010-B0015(on pSB4C5) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451;
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</div>
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<div class="step-recipe">
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R0040-B0034-E1010-B0015(on pSB4C5) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451;  
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</div>
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<div class="step-recipe">
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R0040-B0032-E1010-B0015(on pSB2K3) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451;  
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</div>
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R0040-B0034-E1010-B0015(on pSB2K3) Ex_F Tm:70.4&#8451; & mRFP_R Tm:76.0&#8451;
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</div>
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<div class="step-recipe">
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Kapa-Taq
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R0040-B0032-E1010-B0015(on pSB4C5) R0040-B0034-E1010-B0015(on pSB4C5) R0040-B0032-E1010-B0015(on pSB2K3) R0040-B0034-E1010-B0015(on B2K3)
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R0040-B0032-E1010-B0015(on pSB4C5)
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</div>
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<div class="step-recipe">
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R0040-B0034-E1010-B0015(on pSB4C5)  
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</div>
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<div class="step-recipe">
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R0040-B0032-E1010-B0015(on pSB2K3)  
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</div>
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<div class="step-recipe">
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R0040-B0034-E1010-B0015(on pSB2K3)
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R0040-B0032-E1010-B0015(on pSB4C5)
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R0040-B0032-E1010-B0015(on pSB4C5)]
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        R0040-B0034-E1010-B0015(on pSB4C5)
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</div>
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        R0040-B0032-E1010-B0015(on pSB2K3)
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<div class="step-recipe">
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        R0040-B0034-E1010-B0015(on pSB2K3)
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R0040-B0034-E1010-B0015(on pSB4C5)
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</div>
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<div class="step-recipe">
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R0040-B0032-E1010-B0015(on pSB2K3)
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</div>
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<div class="step-recipe">
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R0040-B0034-E1010-B0015(on pSB2K3)
</div>
</div>
    </div>
    </div>

Revision as of 03:02, 18 October 2014

Notebook
Lab Documents

E1010 Target gene vector exchange to pSB6A1

  • Start

  • PCR

    R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert)
    KOD plus Neo

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)
    Cut insert with EcoRI, PstI (using 10×M buffer)

  • Gel extraction

    digested vector & insert

  • Dephosphorylation

    Antarctic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products
    each of 5.0µL to DH5α

  • PCR

    R0040-B0034-E1010-B0015(on 6A1) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R Tm:72.6℃ (insert) KOD plus Neo

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)
    Cut insert with EcoRI, PstI (using 10×M buffer)

  • Gel extraction

    digested insert

  • Ethanol Precipitation

    digested insert

  • Digestion

    Cut vector with EcoRI, PstI (using 10×H buffer)

  • Dephosphorylation

    Antactic phosphorylate vector (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products
    5.0µL DNA to DH5α

  • Colony PCR

    R0040-B0032-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0034-E1010-B0015(on pSB4C5) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0032-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    R0040-B0034-E1010-B0015(on pSB2K3) Ex_F Tm:70.4℃ & mRFP_R Tm:76.0℃
    Kapa-Taq

  • Liquid Culture

    R0040-B0032-E1010-B0015(on pSB4C5)
    R0040-B0034-E1010-B0015(on pSB4C5)
    R0040-B0032-E1010-B0015(on pSB2K3)
    R0040-B0034-E1010-B0015(on pSB2K3)

  • Mini-Prep

    R0040-B0032-E1010-B0015(on pSB4C5)]
    R0040-B0034-E1010-B0015(on pSB4C5)
    R0040-B0032-E1010-B0015(on pSB2K3)
    R0040-B0034-E1010-B0015(on pSB2K3)

  • Complete!

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