Team:HokkaidoU Japan/Notebook/Length/as90 120

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      Notebook
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<ul style="margin-left:20px;">
<ul style="margin-left:20px;">
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Overview</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Introduction">Introduction</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
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<ul>
<ul>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
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            <li class="ldd_contents"><a href="">Overview</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Overview</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li>
                                                             <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li>
                                                             <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/Outreach/Survey#Education">Education</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
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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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R0040-B0034-E1010-B0015 as RBS XhoI, 90bp as mRFP Nco as RBS XhoI, 120bp as mRFP Nco
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R0040-B0034-E1010-B0015
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</div>
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<div class="step-recipe">
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as_RBS_XhoI_F Tm:68.2&#8451; & 90bp_asmRFP_Nco_R Tm:79.9&#8451;
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</div>
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<div class="step-recipe">
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as_RBS_XhoI_F Tm:68.2&#8451; & 120bp_asmRFP_Nco_R Tm:66.9&#8451;
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      <div class="step-recipe">
      <div class="step-recipe">
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Cut as 90, as120, H-stem with NcoI, XhoI(using 10&times;Cut Smart)
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Cut as90(on H-stem) with NcoI,XhoI(using 10&times;Cut Smart)
 +
</div>
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<div class="step-recipe">
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        Cut as120(on H-stem) with NcoI,XhoI(using 10&times;Cut Smart)
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Dephophorylation
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Dephosphorylation
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H-stem
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Antactic phosphorylate H-stem (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
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Ligate as90, as120 with H-stem
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Ligate as90, as120 with H-stem(on pSB1A3)
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as90, as120 on H-stem 5&micro;L DNA to DH5&alpha; Turbo
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as90, as120(on pSB1A3)
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<div class="step-recipe">
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5&micro;L DNA to DH5&alpha; Turbo
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as90, as120 on H-stem
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as90 (on pSB1A3) XhoI_RBS_ Tm:68.2&#8451; & as90_NcoI_ Tm:79.9&#8451;
</div>
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<div class="step-recipe">
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        as120(on pSB1A3) XhoI_RBS_ Tm:68.2&#8451; & as120_NcoI_ Tm:66.9&#8451;
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<div class="step-recipe">
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        Kapa-Taq
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as90, as120 on H-stem
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R0010-K1524104-B0015(on pSB1A3)
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</div>
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<div class="step-recipe">
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        R0010-K1524105-B0015(on pSB1A3)
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as90, as120 on H-stem
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R0010-K1524104-B0015(on pSB1A3)
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</div>
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<div class="step-recipe">
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        R0010-K1524105-B0015(on pSB1A3)
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as90, as120 on H-stem
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R0010-K1524104-B0015(on pSB1A3)
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</div>
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<div class="step-recipe">
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        R0010-K1524105-B0015(on pSB1A3)
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{{Team:HokkaidoU_Japan/JS}}
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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!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Length/as90_120"