Team:HokkaidoU Japan/Notebook/H Stem/Construction

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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>
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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_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>
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mini-Prep
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Mini-Prep
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      </ul>
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1C3 Vector
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pSB1C3 vector
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      </ul>
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Plac (plasmid) with 100up_F & 200dn_R, Ptet-B0034-mRFP-dT (plasmid) with XhoI-RBS & NcoI-mRFP (anti-sense), double-Terminator (plasmid) with 100up & 200dn
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R0010 (plasmid) with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
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        R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0&#8451; & XhoI_RBS_R Tm:68.2&#8451;
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<div class="step-recipe">
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        B0015 (plasmid) with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
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      </ul>
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Plac with SpeI, anti-sense with NcoI, anti-sense with XhoI, dT with XbaI
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R0010 with SpeI
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        anti-sense with NcoI, XhoI
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        B0015 with XbaI
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      </ul>
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stem_upstream_F & stem_upstream_R, stem_downstream_F & stem_downstream_R
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stem_upstream_F Tm:71.4&#8451; & stem_upstream_R Tm:71.4&#8451;
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stem_downstream_F Tm:71.4&#8451; & stem_downstream_R Tm:71.4&#8451;
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Plac & stem-up & as-up, as-dn & stem-dn & dT
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R0010 & stem-up & as-up  
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<div class="step-recipe">
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as-dn & stem-dn & B0015
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      </ul>
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Plac-stem-as with 100up_F & XhoI_RBS_R, as-stem-dT with NcoI_mRFP_F & 200dn_R
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R0010-stem-as with 100up_F Tm:62.6&#8451; & XhoI_RBS_R Tm:68.2&#8451;
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<div class="step-recipe">
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as-stem-B0015 with NcoI-mRFP_F Tm:76.0&#8451; & 200dn Tm:66.6&#8451;
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      </ul>
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Plac-stem-as & as-stem-dT with 100up_F & 200dn_R
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R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
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      </ul>
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Plac-stem-as-stem-dT with EcoRI & PstI, 1C3 with EcoRI & PstI
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Cut R0010-stem-as-stem-B0015 with EcoRI & PstI
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Cut pSB1C3 with EcoRI & PstI
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      </ul>
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Plac-stem-as-stem-dT & 1C3
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Ligate R0010-stem-as-stem-B0015 with pSB1C3
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Plac-stem-as-stem-dT on 1C3
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R0010-stem-as-stem-B0015(on pSB1C3)
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5.0&micro;L to DH5alpha;
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Plac-stem-as-stem-dT on 1C3 with stem_up_F & stem_d_R / 100up_F & 200dn_R / EX_F & PS_R
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R0010-stem-as-stem-B0015(on pSB1C3) with  
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stem_up_F Tm:71.4&#8451; & stem_dn_R Tm:71.4&#8451;
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100up_F Tm:62.6&#8451; & 200dn_R Tm:66.6&#8451;
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<div class="step-recipe">
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EX_F Tm:70.4&#8451; & PS_R Tm:74.6&#8451;
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Latest revision as of 15:28, 9 September 2015

Notebook
Lab Documents

H-Stem Construction

  • Start

  • Mini-Prep

    pSB1C3 vector

  • PCR

    R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃
    B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    R0010 with SpeI
    anti-sense with NcoI, XhoI
    B0015 with XbaI

  • Primer Annealing

    stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃
    stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃

  • Ligation

    R0010 & stem-up & as-up
    as-dn & stem-dn & B0015

  • PCR

    R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃
    as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃

  • PCR

    R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    Cut R0010-stem-as-stem-B0015 with EcoRI & PstI
    Cut pSB1C3 with EcoRI & PstI

  • Ligation

    Ligate R0010-stem-as-stem-B0015 with pSB1C3

  • Transformation

    R0010-stem-as-stem-B0015(on pSB1C3)
    5.0µL to DH5alpha;

  • Colony PCR

    R0010-stem-as-stem-B0015(on pSB1C3) with
    stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃
    100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    EX_F Tm:70.4℃ & PS_R Tm:74.6℃

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/H_Stem/Construction"