Team:HokkaidoU Japan/Notebook/Pre experiment/Plac-failed-experiment

From 2014.igem.org

(Difference between revisions)
 
(12 intermediate revisions not shown)
Line 1: Line 1:
{{Team:HokkaidoU_Japan/CSS}}
{{Team:HokkaidoU_Japan/CSS}}
 +
{{Team:HokkaidoU_Japan/Top_Header/CSS}}
 +
{{Team:HokkaidoU_Japan/Footer/CSS}}
{{Team:HokkaidoU_Japan/Notebook/CSS}}
{{Team:HokkaidoU_Japan/Notebook/CSS}}
-
 
+
{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}
<html>
<html>
 +
<link href='http://fonts.googleapis.com/css?family=Merriweather' rel='stylesheet' type='text/css'>
 +
<div id="background">
 +
<!--begin header-->
 +
<div id="header-top">
 +
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_Header_Top.png">
 +
</div>
 +
<div id="header-bottom">
 +
  <div id="header-bottom-contents">
 +
    <div class="header-title">
 +
      <div class="header-title-top">
 +
      Notebook
 +
      </div>
 +
      <div class="header-title-bottom">
 +
      Lab Documents
 +
      </div>
 +
    </div>
 +
    <div class="header-picture">
 +
      <img src="https://static.igem.org/mediawiki/2014/4/44/HokkaidoU_Header_ki.png">
 +
    </div>
 +
  </div>
 +
</div>
 +
<!--end header-->
 +
<!--begin menu-->
 +
    <div class="box">
 +
  <div id="box-wrapper">
 +
 +
      <ul id="ldd_menu" class="ldd_menu">
 +
                                <li>
 +
                                <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-->
 +
                                </li>
 +
<li class="menu_button">
 +
<span class="menu-list"><span style="position:relative; top:10px;">RNA in Love</span></span><!-- Increases to 510px in width-->
 +
<div class="ldd_submenu" style="left:-415px; width:970px;">
 +
<ul style="margin-left:20px;">
 +
<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>
 +
</ul>
 +
                                        </div>
 +
 +
</li>
 +
 
 +
</ul>
 +
    </div>
 +
</div>
 +
 +
<!--end box-->
 +
<!-- end menu-->
<div class="wrapper">
<div class="wrapper">
   <div id="hokkaidou-contents">
   <div id="hokkaidou-contents">
Line 50: Line 158:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0010-B0034-E1010-B0015(pSB6A1) from destribution kit 1&micro;L DNA to JM109
+
R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1&micro;L to JM109
</div>
</div>
    </div>
    </div>
Line 71: Line 179:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
pSB6A1
+
R0010-B0034-E1010-B0015(on pSB6A1)
</div>
</div>
    </div>
    </div>
Line 92: Line 200:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
pSB6A1
+
R0010-B0034-E1010-B0015(on pSB6A1)
</div>
</div>
    </div>
    </div>
Line 113: Line 221:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0040-B0034-E1010-B0015 as RBS XhoI, as mRFP NcoI
+
R0040-B0034-E1010-B0015 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>
    </div>
Line 134: Line 245:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP)
+
asmRFP
</div>
</div>
    </div>
    </div>
Line 155: Line 266:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart) Cut anti-sense(mRFP) 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 163: Line 277:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Gel Extraction & Ethanol precipetation
+
Gel Extraction
</h2>
</h2>
  </div>
  </div>
Line 176: Line 290:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
pHN1257 anti-sense(mRFP)
+
pHN1257 & asmRFP
</div>
</div>
    </div>
    </div>
  </div>
  </div>
</li>
</li>
 +
<li class="step">
 +
  <div class="step-tile">
 +
  <h2 class="step-title">
 +
Ethanol precipetation
 +
</h2>
 +
  </div>
 +
  <div class="step-detail">
 +
    <div class="step-detail-body">
 +
      <ul class="step-detail-trials">
 +
        <li>
 +
  <time>
 +
Sun Jul 27
 +
</time>
 +
</li>
 +
      </ul>
 +
      <div class="step-recipe">
 +
pHN1257 & asmRFP
 +
</div>
 +
    </div>
 +
  </div>
 +
</li>
 +
<li class="step">
<li class="step">
  <div class="step-tile">
  <div class="step-tile">
Line 197: Line 333:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ligate anti-sense(mFP) with pHN1257
+
Ligate asmRFP with pHN1257
</div>
</div>
    </div>
    </div>
Line 218: Line 354:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257 5&micro;L DNA to DH5&alpha; Turbo
+
asmRFP(on pHN1257)
 +
</div>
 +
<div class="step-recipe">
 +
5&micro;L to DH5&alpha; Turbo
</div>
</div>
    </div>
    </div>
Line 239: Line 378:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257
+
asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
Kapa-Taq
</div>
</div>
    </div>
    </div>
Line 260: Line 402:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257
+
asmRFP(on pHN1257)
</div>
</div>
    </div>
    </div>
Line 281: Line 423:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257
+
asmRFP(on pHN1257)
</div>
</div>
    </div>
    </div>
Line 289: Line 431:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
Transformation
+
DoubleTransformation
</h2>
</h2>
  </div>
  </div>
Line 302: Line 444:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
anti-sense(mRFP) on pHN1257 & pSB6A1 2.5&micro;L each DNA to DH5&alpha; Turbo
+
asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
 +
</div>
 +
<div class="step-recipe">
 +
2.5&micro;L each to DH5&alpha; Turbo
</div>
</div>
    </div>
    </div>
Line 323: Line 468:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Culture anti-sense(mRFP) on pHN1257 & pSB6A1 1. 2mL LB with 100&micro;L 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.
+
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
 +
</div>
 +
<div class="step-recipe">
 +
1. 2mL LB with 100&micro;L 100mM IPTG  
 +
2. 2mL LB However, IPTG was too much to grow normally.
 +
 
</div>
</div>
    </div>
    </div>
Line 344: Line 494:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Culture anti-sense(mRFP) on pHN1257 & pSB6A1 E. coli was early growth 1. 2mL LB with 20&micro;L 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.
+
Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20&micro;L 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.
 +
</div>
</div>
    </div>
    </div>
Line 354: Line 505:
   </ul>
   </ul>
</div>
</div>
 +
</div>
 +
</div>
 +
<!--begin footer-->
 +
 +
<div id="footer-wrapper" style="background-color: #d92424;">
 +
  <div id="footer-content">
 +
<div id="footer-logo">
 +
<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 id="footer-twitter">
 +
        <a href="http://twitter.com/igem_hokkaidou"><img style="height:150px; position:relative; bottom:0;" src="https://static.igem.org/mediawiki/2014/3/3b/HokkaidoU_Footer_Twitter.png"></a>
 +
    </div>
 +
 +
 +
  </div>
 +
</div>
 +
</div><!--background-->
 +
<link href='http://fonts.googleapis.com/css?family=Special+Elite' rel='stylesheet' type='text/css'>
 +
</html>
</html>
 +
{{Team:HokkaidoU_Japan/JS}}

Latest revision as of 15:25, 9 September 2015

Notebook
Lab Documents

Wrong Plac failed Experiment

  • Start

  • Get anti-sense vector

    pHN1257 Great thanks to N. Nakashima

  • Transformation

    R0010-B0034-E1010-B0015(on pSB6A1) from destribution kit 1µL to JM109

  • Liquid Culture

    R0010-B0034-E1010-B0015(on pSB6A1)

  • Mini-prep

    R0010-B0034-E1010-B0015(on pSB6A1)

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76/0℃
    KOD plus Neo

  • Ehanol precipetation

    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 precipetation

    pHN1257 & asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-prep

    asmRFP(on pHN1257)

  • DoubleTransformation

    asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    2.5µL each to DH5α Turbo

  • Asssay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1)
    1. 2mL LB with 100µL 100mM IPTG 2. 2mL LB However, IPTG was too much to grow normally.

  • Assay(unsuccess)

    Culture asmRFP(on pHN1257) & B0034-E1010-B0015(on pSB6A1) E. coli was early growth 1. 2mL LB with 20µL 100mM IPTG 2. 2mL LB However, pLac was inducible promoter that is promoted by IPTG. We decided to retry this examination.

  • Failure...

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/Plac-failed-experiment"