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/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_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li>
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                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</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_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</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">Length Variation</a></li>
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            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">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#Conclusion">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/Notebook">Notebook</a></li>
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   <div id="hokkaidou-contents">
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   <h2>
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Stem region replacement
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Stem Region Replacement
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pHN1257
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pHN1257 stem_F Tm:70.6&#8451;  terminator_R Tm:71.4&#8451;
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KOD FX NEO
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R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
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R0010  
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each of 5.0µL DNA to DH5α  
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Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)
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Cut R0010 with PstI, SpeI (using 10xH buffer)  
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Cut pHN1257 with XbaI, PstI (using 10xM buffer)
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Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
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Ligation products  
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each of 5.0µL DNA to DH5α
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R0010-stem-dT 100up f 162primer R(あとで書く)
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R0010-stem-B0015 with 100up_F pHN1257terminator_R
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R0010-stem-dT 100up 162 KOD FX NEO
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R0010-stem-dT with 100up_F pHN1257terminator_R
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KOD FX NEO
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{{Team:HokkaidoU_Japan/JS}}

Latest revision as of 15:26, 9 September 2015

Notebook
Lab Documents

Stem Region Replacement

  • Start

  • PCR

    pHN1257 stem_F Tm:70.6℃ terminator_R Tm:71.4℃
    KOD FX NEO

  • Transformation

    R0010
    each of 5.0µL DNA to DH5α

  • Digestion

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

  • Ligation

    Ligate R0010 with pHN1257

  • Transformation

    Ligation products
    each of 5.0µL DNA to DH5α

  • Colony PCR

    R0010-stem-B0015 with 100up_F pHN1257terminator_R

  • Mini-Prep

    R0010-stem-B0015

  • PCR(confirm)

    R0010-stem-dT with 100up_F pHN1257terminator_R
    KOD FX NEO

  • Complete!

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