Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

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{{Team:HokkaidoU_Japan/CSS}}
{{Team:HokkaidoU_Japan/CSS}}
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{{Team:HokkaidoU_Japan/Top_Header/CSS}}
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{{Team:HokkaidoU_Japan/Footer/CSS}}
{{Team:HokkaidoU_Japan/Notebook/CSS}}
{{Team:HokkaidoU_Japan/Notebook/CSS}}
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{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}
<html>
<html>
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<link href='http://fonts.googleapis.com/css?family=Merriweather' rel='stylesheet' type='text/css'>
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<div id="background">
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<!--begin header-->
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<div id="header-top">
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<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_Header_Top.png">
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</div>
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<div id="header-bottom">
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  <div id="header-bottom-contents">
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    <div class="header-title">
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      <div class="header-title-top">
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      Notebook
 +
      </div>
 +
      <div class="header-title-bottom">
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      Lab Documents
 +
      </div>
 +
    </div>
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    <div class="header-picture">
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      <img src="https://static.igem.org/mediawiki/2014/4/44/HokkaidoU_Header_ki.png">
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    </div>
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  </div>
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</div>
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<!--end header-->
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<!--begin menu-->
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    <div class="box">
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  <div id="box-wrapper">
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 +
      <ul id="ldd_menu" class="ldd_menu">
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                                <li>
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                                <span class="menu-list" ><a href="https://2014.igem.org/Team:HokkaidoU_Japan"><span style="position:relative; top:10px;" >Top</span></a></span><!-- Increases to 510px in width-->
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                                </li>
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<li class="menu_button">
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<span class="menu-list"><span style="position:relative; top:10px;">RNA in Love</span></span><!-- Increases to 510px in width-->
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<div class="ldd_submenu" style="left:-415px; width:970px;">
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<ul style="margin-left:20px;">
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</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>
 +
</ul>
 +
<ul>
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li>
 +
                                                        <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li>
 +
</ul>
 +
<ul>
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li>
 +
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li>
 +
</ul>
 +
<ul>
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li>
 +
            <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#Results">Results</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li>
 +
</ul>
 +
<ul>
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li>
 +
            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li>
 +
                                                            <li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li>
 +
</ul>
 +
</div>
 +
</li>
 +
<li class="menu_button">
 +
<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span>
 +
<div class="ldd_submenu" style="width:590px; left:-230px;">
 +
<ul style="margin-left:20px;">
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li>
 +
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li>
 +
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li>
 +
</ul>
 +
<ul>
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High School</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>
 +
</ul>
 +
<ul>
 +
<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#Evaluation">Evaluation</a></li>
 +
</ul>
 +
</div>
 +
</li>
 +
<li class="menu_button">
 +
<span class="menu-list"><span style="position:relative; top:10px;">About Us</span></span>
 +
<div class="ldd_submenu" style="width:204px; left: -30px;">
 +
<ul style="margin-left:20px;">
 +
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li>
 +
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li>
 +
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li>
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</ul>
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                                        </div>
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</li>
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 +
</ul>
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    </div>
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</div>
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<!--end box-->
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<!-- end menu-->
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<div class="wrapper">
<div class="wrapper">
   <div id="hokkaidou-contents">
   <div id="hokkaidou-contents">
 +
  <h2>
 +
Anti-Sense mRFP Construction
 +
</h2>
     <div class="workflow-container">
     <div class="workflow-container">
       <ul class="steps">
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  <h2>Start</h2>
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  <h2 class="step-title">
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Primer Annealing
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Get anti-sense vector
</h2>
</h2>
  </div>
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-
  <div class="stap-detail">
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    <div class="step-detail-body">
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  <time>
  <time>
-
Sun Aug 17
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Fri Jun 20
</time>
</time>
</li>
</li>
      </ul>
      </ul>
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      <div class="step-recipi">
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      <div class="step-recipe">
-
asB0034-f Tm: 68.7&deg;C asB0034-r Tm: 68.7&deg;C
+
Great thanks to N. Nakashima
</div>
</div>
    </div>
    </div>
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PCR & PCR purification
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  <div class="step-detail">
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  <time>
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Fri Aug 15
 +
</time>
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</li>
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      </ul>
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      <div class="step-recipe">
 +
R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;
 +
</div>
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<div class="step-recipe">
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KOD plus Neo
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</li>
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Ethanol Precipitation
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</h2>
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  <div class="step-detail">
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  <time>
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Fri Aug 15
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</time>
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</li>
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      </ul>
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      <div class="step-recipe">
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asmRFP
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</div>
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    </div>
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  </div>
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</li>
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<li class="step">
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  <div class="step-tile">
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  <h2 class="step-title">
Digestion
Digestion
</h2>
</h2>
  </div>
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Sun Aug 17
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Fri Aug 15
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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)
 +
</div>
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<div class="step-recipe">
 +
Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
</div>
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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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      <div class="step-recipi">
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digested pHN1257
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pHN1257 asmRFP
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Ethanol Precipitation
Ethanol Precipitation
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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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Ligate pHN1257 with asB0034
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asmRFP
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Ligation
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Tue Aug 19
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Sat Aug 16
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Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0&micro;L DNA to DH5&alpha; 42&deg;C        1min on ice 2min
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Ligate asmRFP with pHN1257
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Transformation
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Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1 asB0034-R Tm:68.7
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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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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>
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Kapa-Taq
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Liquid Culture
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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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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; 42C    30sec on ice  2min
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asmRFP(pHN1257)
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{{Team:HokkaidoU_Japan/JS}}

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"