Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement

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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_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>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li>
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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#Overview">Overview</a></li>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Project/H_Stem#Modeling">Modelling</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_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/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/asB0034/Conclusion">Conclusion</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Overview">Length Variation</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length/Overview">Overview</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/Conclusiton">Conclusion</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li>
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                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li>
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                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li>
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<span class="menu-list">Policy and Practice</span>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Education">Education</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Education#School_Festival">School Festival</a></li>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion">Discussion</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion#Background">Background</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Policy_And_Practice/Discussion#Estimation">Estimation</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li>
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{{Team:HokkaidoU_Japan/JS}}

Revision as of 11:25, 14 October 2014

Stem Region Replacement

  • Start

  • PCR

    pHN1257 161 Tm: 162 Tm: KOD FX NEO

  • Transformation

    R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min

  • Digestion

    Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)

  • Ligation

    Ligate R0010 with pHN1257

  • Transformation

    Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min

  • Colony PCR

    R0010-stem-dT 100up f 162primer R(あとで書く)

  • mini-Prep

    R0010-stem-dT

  • PCR(confirm)

    R0010-stem-dT 100up 162 KOD FX NEO

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/Stem_replacement"