Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement

From 2014.igem.org

(Difference between revisions)
 
(22 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">
   <h2>
   <h2>
-
Stem region replacement
+
Stem Region Replacement
</h2>
</h2>
     <div class="workflow-container">
     <div class="workflow-container">
       <ul class="steps">
       <ul class="steps">
         <li class="step start">
         <li class="step start">
-
  <div class="step-title">
+
  <div class="step-tile">
  <h2 class="step-title">Start</h2>
  <h2 class="step-title">Start</h2>
  </div>
  </div>
Line 30: Line 141:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
pHN1257
+
pHN1257 stem_F Tm:70.6&#8451;  terminator_R Tm:71.4&#8451;
 +
</div>
 +
<div class="step-recipe">
 +
KOD FX NEO
</div>
</div>
    </div>
    </div>
Line 51: Line 165:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
+
R0010  
 +
</div>
 +
<div class="step-recipe">
 +
each of 5.0µL DNA to DH5α  
</div>
</div>
    </div>
    </div>
Line 72: Line 189:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)
+
Cut R0010 with PstI, SpeI (using 10xH buffer)  
 +
</div>
 +
<div class="step-recipe">
 +
Cut pHN1257 with XbaI, PstI (using 10xM buffer)
</div>
</div>
    </div>
    </div>
Line 114: Line 234:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min
+
Ligation products  
 +
</div>
 +
<div class="step-recipe">
 +
each of 5.0µL DNA to DH5α
</div>
</div>
    </div>
    </div>
Line 135: Line 258:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0010-stem-dT 100up f 162primer R(あとで書く)
+
R0010-stem-B0015 with 100up_F pHN1257terminator_R
</div>
</div>
    </div>
    </div>
Line 143: Line 266:
  <div class="step-tile">
  <div class="step-tile">
  <h2 class="step-title">
  <h2 class="step-title">
-
mini-Prep
+
Mini-Prep
</h2>
</h2>
  </div>
  </div>
Line 156: Line 279:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0010-stem-dT
+
R0010-stem-B0015
</div>
</div>
    </div>
    </div>
Line 177: Line 300:
      </ul>
      </ul>
      <div class="step-recipe">
      <div class="step-recipe">
-
R0010-stem-dT 100up 162 KOD FX NEO
+
R0010-stem-dT with 100up_F pHN1257terminator_R
 +
</div>
 +
<div class="step-recipe">
 +
KOD FX NEO
</div>
</div>
    </div>
    </div>
Line 187: Line 313:
   </ul>
   </ul>
</div>
</div>
-
<html>
+
</div>
 +
</div>
 +
<!--begin footer-->
 +
<div id="footer-wrapper" style="background-color:#25928E;">
 +
<!--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>
 +
{{Team:HokkaidoU_Japan/JS}}

Latest revision as of 15:26, 9 September 2015

Notebook
Lab Documents

Stem Region Replacement

  • Start

  • PCR

    pHN1257 stem_F Tm:70.6℃ terminator_R Tm:71.4℃
    KOD FX NEO

  • Transformation

    R0010
    each of 5.0µL DNA to DH5α

  • Digestion

    Cut R0010 with PstI, SpeI (using 10xH buffer)
    Cut pHN1257 with XbaI, PstI (using 10xM buffer)

  • Ligation

    Ligate R0010 with pHN1257

  • Transformation

    Ligation products
    each of 5.0µL DNA to DH5α

  • Colony PCR

    R0010-stem-B0015 with 100up_F pHN1257terminator_R

  • Mini-Prep

    R0010-stem-B0015

  • PCR(confirm)

    R0010-stem-dT with 100up_F pHN1257terminator_R
    KOD FX NEO

  • Complete!

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