Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

From 2014.igem.org

(Difference between revisions)
 
(7 intermediate revisions not shown)
Line 29: Line 29:
<!--begin menu-->
<!--begin menu-->
     <div class="box">
     <div class="box">
-
   <div id="box-wrapper">  
+
   <div id="box-wrapper">
       <ul id="ldd_menu" class="ldd_menu">
       <ul id="ldd_menu" class="ldd_menu">
Line 82: Line 82:
</ul>
</ul>
<ul>
<ul>
-
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li>
+
<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#Education">Education</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li>
Line 89: Line 89:
<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>
-
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li>
+
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Evaluation">Evaluation</a></li>
</ul>
</ul>
</div>
</div>
Line 102: Line 102:
</ul>
</ul>
                                         </div>
                                         </div>
-
+
 
</li>
</li>
-
 
+
 
</ul>
</ul>
     </div>
     </div>
Line 114: Line 114:
<div class="wrapper">
<div class="wrapper">
   <div id="hokkaidou-contents">
   <div id="hokkaidou-contents">
-
<h2>anti-sense mRFP Construction</h2>
+
  <h2>
 +
Anti-Sense mRFP Construction
 +
</h2>
     <div class="workflow-container">
     <div class="workflow-container">
       <ul class="steps">
       <ul class="steps">
Line 125: Line 127:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Primer Annealing
+
Get anti-sense vector
</h2>
</h2>
  </div>
  </div>
Line 133: Line 135:
        <li>
        <li>
  <time>
  <time>
-
Sun Aug 17
+
Fri Jun 20
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
asB0034_F Tm: 68.7&#8451; asB0034_R Tm: 68.7&#8451;
+
Great thanks to N. Nakashima
 +
</div>
 +
    </div>
 +
  </div>
 +
</li>
 +
<li class="step">
 +
  <div class="step-tile">
 +
  <h2 class="step-title">
 +
PCR & PCR purification
 +
</h2>
 +
  </div>
 +
  <div class="step-detail">
 +
    <div class="step-detail-body">
 +
      <ul class="step-detail-trials">
 +
        <li>
 +
  <time>
 +
Fri Aug 15
 +
</time>
 +
</li>
 +
      </ul>
 +
      <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>
 +
<div class="step-recipe">
 +
KOD plus Neo
 +
</div>
 +
    </div>
 +
  </div>
 +
</li>
 +
<li class="step">
 +
  <div class="step-tile">
 +
  <h2 class="step-title">
 +
Ethanol Precipitation
 +
</h2>
 +
  </div>
 +
  <div class="step-detail">
 +
    <div class="step-detail-body">
 +
      <ul class="step-detail-trials">
 +
        <li>
 +
  <time>
 +
Fri Aug 15
 +
</time>
 +
</li>
 +
      </ul>
 +
      <div class="step-recipe">
 +
asmRFP
</div>
</div>
    </div>
    </div>
Line 154: Line 201:
        <li>
        <li>
  <time>
  <time>
-
Sun Aug 17
+
Fri Aug 15
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut pHN1257 with NcoI, XhoI(using 10&times;cut smart)
+
Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)
 +
</div>
 +
<div class="step-recipe">
 +
Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
</div>
</div>
    </div>
    </div>
Line 167: Line 217:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Gel extraction
+
Gel Extraction
</h2>
</h2>
  </div>
  </div>
Line 175: Line 225:
        <li>
        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
digested pHN1257
+
pHN1257 asmRFP
</div>
</div>
    </div>
    </div>
Line 196: Line 246:
        <li>
        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
digested pHN1257
+
asmRFP
</div>
</div>
    </div>
    </div>
Line 209: Line 259:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Ligation
+
Dephosphorylation
</h2>
</h2>
  </div>
  </div>
Line 217: Line 267:
        <li>
        <li>
  <time>
  <time>
-
Mon Aug 18
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ligate pHN1257 with asB0034
+
asmRFP
</div>
</div>
    </div>
    </div>
Line 230: Line 280:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Transformation
+
Ligation
</h2>
</h2>
  </div>
  </div>
Line 238: Line 288:
        <li>
        <li>
  <time>
  <time>
-
Tue Aug 19
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0&micro;L DNA to DH5&alpha; 42&#8451;        1min on ice 2min
+
Ligate asmRFP with pHN1257
</div>
</div>
    </div>
    </div>
Line 251: Line 301:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Colony PCR
+
Transformation
</h2>
</h2>
  </div>
  </div>
Line 259: Line 309:
        <li>
        <li>
  <time>
  <time>
-
Wed Aug 20
+
Sat Aug 16
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1&#8451; asB0034_R Tm:68.7&#8451;
+
asmRFP(on pHN1257)  
 +
</div>
 +
<div class="step-recipe">
 +
5&micro;L to DH5&alpha; Turbo
</div>
</div>
    </div>
    </div>
Line 272: Line 325:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Culture
+
Colony PCR
</h2>
</h2>
  </div>
  </div>
Line 280: Line 333:
        <li>
        <li>
  <time>
  <time>
-
Thu Aug 21
+
Sun Aug 17, Mon Aug 18
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem
+
asmRFP(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & pHN1257_terminator_R Tm:71.4&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
Kapa-Taq
</div>
</div>
    </div>
    </div>
Line 293: Line 349:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
mini-Prep
+
Liquid Culture
</h2>
</h2>
  </div>
  </div>
Line 301: Line 357:
        <li>
        <li>
  <time>
  <time>
-
Fri Aug 22
+
Mon Aug 18
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ptrc-stem-asB0034-stem
+
asmRFP(on pHN1257)
</div>
</div>
    </div>
    </div>
Line 314: Line 370:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Transformation
+
Mini-Prep
</h2>
</h2>
  </div>
  </div>
Line 322: Line 378:
        <li>
        <li>
  <time>
  <time>
-
Fri Aug 22
+
Tue Aug 19
</time>
</time>
</li>
</li>
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
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
+
asmRFP(pHN1257)
</div>
</div>
    </div>
    </div>
Line 346: Line 402:
   <div id="footer-content">
   <div id="footer-content">
<div id="footer-logo">
<div id="footer-logo">
-
<a href="http://igemhokkaidou.com/"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a>
+
<a href="http://igemhokkaidou.wordpress.com"><img style="height:150px;position:relative;" src="https://static.igem.org/mediawiki/2014/3/39/HokkaidoU_logo_transparent.png"></a>
</div>
</div>
<div id="footer-twitter">
<div id="footer-twitter">

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"