Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT

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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_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_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/Results">Results</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Method">Method</a></li>
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                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</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>
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                                                             <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/Conclusiton">Conclusion</a></li>
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   <div id="hokkaidou-contents">
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   <h2 class="step-title">P-tet-B0032-mRFP-pdT</h2>
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   <h2 class="step-title">Ptet-B0032-mRFP-dT</h2>
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      </ul>
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Ptet-B0034-mRFP-dT(6A1) SpeI-PstI-F& Ptet-SpeI-R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo
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R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8&#8451;& Ptet_SpeI_R Tm:72.6&#8451; (vector)
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</div>
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<div class="step-recipe">
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XbaI_B0032_mRFP_F Tm:76.3&#8451; & PstI_SpeI_R Tm:74.6&#8451; (insert)
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<div class="step-recipe">
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KOD Fx Neo
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digested PCR productions
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PCR products
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Cut  vector with SpeI, PstI (using 10&times;H buffer) Cut  insert with XbaI, PstI (using 10&times;M buffer)
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Cut  vector with SpeI, 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 XbaI, PstI (using 10&times;M buffer)
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Antarctic phosphatase
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Dephosphorylation
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Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&deg;C 35min 65&deg;C 10min
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Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
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ligation products on ice  30min each of 5.0&micro;L DNA to DH5&alpha;  42&deg;C        1min on ice 2min
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R0040-B0032-E1010-B0015(on pSB1A6)
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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;   
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Ptet-B0034-mRFP-dT on pSB6A1 EcoI-XbaI-F Tm:70.9 rbs-SpeI-R Tm:59.2
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R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9&#8451; & RBS_SpeI_R Tm:59.2&#8451;
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Ptet-B0034-mRFP-dT on pSB6A1
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R0040-B0032-E1010-B0015(on pSB6A1)
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mini-Prep
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Mini-Prep
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Ptet-B0034-mRFP-dT on pSB6A1
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R0040-B0032-E1010-B0015(on pSB6A1)
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{{Team:HokkaidoU_Japan/JS}}
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Latest revision as of 15:25, 9 September 2015

Notebook
Lab Documents

Ptet-B0032-mRFP-dT

  • Start

  • PCR

    R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8℃& Ptet_SpeI_R Tm:72.6℃ (vector)
    XbaI_B0032_mRFP_F Tm:76.3℃ & PstI_SpeI_R Tm:74.6℃ (insert)
    KOD Fx Neo

  • Gel extraction

    PCR products

  • Digestion

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

  • Dephosphorylation

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

  • Ligation

    Ligate vector with insert

  • Transformation

    R0040-B0032-E1010-B0015(on pSB1A6)
    5.0µL DNA to DH5α

  • Colony PCR

    R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9℃ & RBS_SpeI_R Tm:59.2℃

  • Culture

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Mini-Prep

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Complete!

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