Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

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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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<h2>anti-sense mRFP Construction</h2>
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  <h2>
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Anti-Sense mRFP Construction
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Primer Annealing
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Get anti-sense vector
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Sun Aug 17
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Fri Jun 20
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asB0034_F Tm: 68.7&#8451; asB0034_R Tm: 68.7&#8451;
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Great thanks to N. Nakashima
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PCR & PCR purification
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  <time>
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Fri Aug 15
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R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;  
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</div>
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<div class="step-recipe">
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KOD plus Neo
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Ethanol Precipitation
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</h2>
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  <time>
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Fri Aug 15
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</time>
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      <div class="step-recipe">
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asmRFP
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Sun Aug 17
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Fri Aug 15
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      <div class="step-recipe">
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Cut pHN1257 with NcoI, XhoI(using 10&times;cut smart)
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)
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</div>
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<div class="step-recipe">
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Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
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Gel extraction
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Gel Extraction
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Mon Aug 18
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Sat Aug 16
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digested pHN1257
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pHN1257 asmRFP
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Mon Aug 18
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Sat Aug 16
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digested pHN1257
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asmRFP
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Ligation
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Dephosphorylation
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Mon Aug 18
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Sat Aug 16
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      <div class="step-recipe">
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Ligate pHN1257 with asB0034
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asmRFP
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Transformation
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Ligation
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Tue Aug 19
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Sat Aug 16
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      <div class="step-recipe">
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Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0&micro;L DNA to DH5&alpha; 42&#8451;        1min on ice 2min
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Ligate asmRFP with pHN1257
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Colony PCR
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Transformation
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Wed Aug 20
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Sat Aug 16
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      <div class="step-recipe">
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Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1&#8451; asB0034_R Tm:68.7&#8451;
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asmRFP(on pHN1257)  
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</div>
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<div class="step-recipe">
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5&micro;L to DH5&alpha; Turbo
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Culture
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Colony PCR
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Thu Aug 21
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Sun Aug 17, Mon Aug 18
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      <div class="step-recipe">
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Ptrc-stem-asB0034-stem
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asmRFP(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & pHN1257_terminator_R Tm:71.4&#8451;
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<div class="step-recipe">
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Kapa-Taq
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mini-Prep
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Liquid Culture
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Fri Aug 22
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Mon Aug 18
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      <div class="step-recipe">
      <div class="step-recipe">
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Ptrc-stem-asB0034-stem
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asmRFP(on pHN1257)
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Transformation
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Mini-Prep
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Tue Aug 19
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Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice  30min each of 2.5&micro;L DNA to DH5&alpha; 42&#8451;    30sec on ice  2min
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asmRFP(pHN1257)
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Latest revision as of 15:23, 9 September 2015

Notebook
Lab Documents

Anti-Sense mRFP Construction

  • Start

  • Get anti-sense vector

    Great thanks to N. Nakashima

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    KOD plus Neo

  • Ethanol Precipitation

    asmRFP

  • Digestion

    Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart)
    Cut asmRFP with NcoI, XhoI (using 10×Cut Smart)

  • Gel Extraction

    pHN1257 asmRFP

  • Ethanol Precipitation

    asmRFP

  • Dephosphorylation

    asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) pHN1257_up_F Tm:60.1℃ & pHN1257_terminator_R Tm:71.4℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-Prep

    asmRFP(pHN1257)

  • Complete!

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