Difference between revisions of "Team:HokkaidoU Japan/Experiments"

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{{HokkaidoU_Japan}}
 
{{HokkaidoU_Japan}}
 
{{Team:HokkaidoU_Japan/CSS}}
 
{{Team:HokkaidoU_Japan/CSS}}
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<html>
 
  
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<br>
 
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<div>
 
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<img src="https://static.igem.org/mediawiki/2016/a/a5/T--HokkaidoU_Japan--Universal_Festival.png"
 
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width="270px" height="90px" alt="Universal_Festival">
 
  
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<span class="nomal2">
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<html>
−
<br>We held a workshop at Hokkaido University Festival from June 2 to June 5, 2016.
+
<head>
−
This event aims to answer questions such as "What is DNA?", "What is Gene Recommbination?" and so on and deepen understanding of DNA and gene recommbination.
+
<style type="text/css">
−
We condctuded a poster session, an easy experiment "Detection of DNA extraction from banana" and crafting, "crafting DNA keyholder".
+
.ka-soru {
 +
cursor: pointer
 +
}
  
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<br>
+
.ichi{
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<br>
+
position: absolute;
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<h3>Experiment</h3>
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left: 30px;
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Participants can see the DNA with their eyes and feel more familiar to DNA.
+
}
  
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<br>
 
−
<br>
 
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<h2>protocol</h2>
 
  
−
<table  style="border-style: none; float: right;" height="280px" width="170px">
+
</style>
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<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--HumanPractice_hokudai_festival22.jpg"width="260px" height="150px" alt="Universal_Festival"></center></td></tr>
+
−
</table>
+
  
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<ol>
+
</head>
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  <li>Crush one third of a banana with a spoon; to break its cell wall.</li>
+
−
  <li>Mix well 10% saltine water 30 mL and detergent 2.5 mL; Saltine water makes nucleic acid and salt. Detergent breaks cell membrane.</li>
+
−
  <li>Pour 1 and 2 into a beaker through coffee filter and filtrate them.</li>
+
−
  <li>After 5 minutes or so, pour 100% ethanol 40 mL into the beaker slowly.</li>
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−
  <li>Then you’ll see some white lump. That’s exactly the DNA!!</li>
+
−
</ol>
+
−
</span>
+
−
<br clear="all">
+
  
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<span class="nomal2">
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<body>
−
<br>
+
−
<br>
+
 
<br>
 
<br>
−
<h2>Crafting</h2>
+
<img src="https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png" alt="Protcols">
−
Participants craft a DNA keyholder and learn the structure of DNA.
+
−
They can take their work home.
+
−
</span>
+
  
 +
<div align="right"><a href="https://2016.igem.org/Team:HokkaidoU_Japan/Notebook"><span class="big">Notebook</span></a></div>
  
−
<br>
 
  
−
<center>
+
<div class="ichi">
−
<table  style="border-style: none;" height="400px" width="400px">
+
−
<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/f/fb/T--HokkaidoU_Japan--HumanPractice_UniFes.jpg"width="400px" height="400px" alt="Universal_Festival">
+
−
</center></td>
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−
<td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/b/bb/T--HokkaidoU_Japan--HumanPractice_hokudai_festival1.jpg"width="400px" height="400px" alt="Universal_Festival">
+
−
</center></td></tr>
+
−
</table>
+
−
</center>
+
  
−
<center><img src="https://static.igem.org/mediawiki/2016/3/30/T--HokkaidoU_Japan--HumanPractice_hokudai_festival2.jpg"width="400px" height="146" alt="Universal_Festival"></center>
 
−
 
−
<span class="nomal2">
 
 
<br>
 
<br>
−
<br>
+
<h1 onClick="hyoji1()"><span class="ka-soru">PCR</span></h1>
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<br>After the activities, we took the survey and got 117 answers</p>
+
−
<br>
+
−
</span>
+
  
−
<table style="border-style: none">
 
−
 
−
<tr align="center" style="border-style: none">
 
−
<td style="border-style: none;"><span class="small">SEX</span></td>
 
−
<td style="border-style: none;"><span class="small">AGE</span></td>
 
−
<td style="border-style: none;"><span class="small">School</span></td>
 
−
</tr>
 
  
−
<tr align="center" style="border-style: none">  
+
<div id="disp1">
−
<td style="border-style: none; ">
+
<span class="nomal2">
−
<img src="https://static.igem.org/mediawiki/2016/d/d0/T--HokkaidoU_Japan--u1.png" style="width:300px; height:250px;" alt="u1">
+
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
−
</td>
+
−
<td style="border-style: none; ">
+
−
<img src="https://static.igem.org/mediawiki/2016/2/28/T--HokkaidoU_Japan--u2.png" style="width:300px; height:250px;" alt="u2">
+
−
</td>
+
−
<td style="border-style: none; ">
+
−
<img src="https://static.igem.org/mediawiki/2016/9/92/T--HokkaidoU_Japan--u3.png" style="width:300px; height:250px;" alt="u3">
+
−
</td>
+
−
</tr>
+
  
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<tr align="center" style="border-style: none">  
+
<table>
−
<td style="border-style: none;"><span class="small"><br>What does "bio-″mean?</span></td>
+
  <tr align="center">
−
<td style="border-style: none;"><span class="small"><br>Do you know iGEM?</span></td>
+
    <th>Solution</th>
−
<td style="border-style: none;"><span class="small"><br>Do you know DNA?</span></td>
+
    <td>template DNA</td>
−
</tr>
+
    <td>Primer-F 10 &micro;M</td>
 +
    <td>Primer-R 10 &micro;M</td>
 +
    <td>MgSO<sub>4</sub></td>
 +
    <td>dNTPs</td>
 +
    <td>10x Buffer</td>
 +
    <td>KOD Plus Neo</td>
 +
    <td>DW</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>1</td>
 +
    <td>1</td>
 +
    <td>1</td>
 +
    <td>3</td>
 +
    <td>5</td>
 +
    <td>5</td>
 +
    <td>1</td>
 +
    <td>33</td>
 +
    <td>50</td>
 +
  </tr>
 +
</table>
 +
Thermal protocol is following
 +
<h2>2STEP Cycle (Tm value &gt; 63&deg;C)</h2>
 +
<table>
 +
  <tr align="center">
 +
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>1</td><td>94</td><td>120</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>2</td><td>98</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>68</td><td>30 / 1kbp</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>4</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycle: sequence 2~3 &times; (25~45)
  
−
<tr align="center" style="border-style: none">  
+
<h2>3STEP Cycle (Tm value &lt; 63&deg;C)</h2>
−
<td style="border-style: none;">
+
<table>
−
<img src="https://static.igem.org/mediawiki/2016/3/3e/T--HokkaidoU_Japan--u4.png" style="width:300px; height:250px;" alt="u4">
+
  <tr align="center">
−
</td>
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
−
<td style="border-style: none;">
+
  </tr>
−
<img src="https://static.igem.org/mediawiki/2016/c/c8/T--HokkaidoU_Japan--u5.png" style="width:300px; height:250px;" alt="u5">
+
  <tr align="center">
−
</td>
+
    <td>1</td><td>94</td><td>120</td>
−
<td style="border-style: none;">
+
  </tr>
−
<img src="https://static.igem.org/mediawiki/2016/4/47/T--HokkaidoU_Japan--u6.png" style="width:300px; height:250px;" alt="u6">
+
  <tr align="center">
−
</td>
+
    <td>2</td><td>98</td><td>10</td>
−
</tr>
+
  </tr>
−
 
+
  <tr align="center">
−
<tr align="center" style="border-style: none">  
+
    <td>3</td><td>Tm</td><td>30</td>
−
<td style="border-style: none;"><span class="small"><br>Did you understand what DNA is?</span></td>
+
  </tr>
−
<td style="border-style: none;"><span class="small"><br>Did you feel familiar to DNA through<br>the experiment or making the accessory?</span></td>
+
  <tr align="center">
−
<td style="border-style: none;"><span class="small"><br>Did you enjoy our programs?</span></td>
+
    <td>4</td><td>68</td><td>30 / 1kbp</td>
−
</tr>
+
  </tr>
 +
  <tr align="center">
 +
    <td>5</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycle: sequence 2~4 &times; (25~45)
  
−
<tr align="center" style="border-style: none">  
+
<h2>STEP DOWN</h2>
−
<td style="border-style: none;">
+
<table>
−
<img src="https://static.igem.org/mediawiki/2016/c/ce/T--HokkaidoU_Japan--u7.png" style="width:300px; height:250px;" alt="u7">
+
  <tr align="center">
−
</td>
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
−
<td style="border-style: none;">
+
  </tr>
−
<img src="https://static.igem.org/mediawiki/2016/2/28/T--HokkaidoU_Japan--u8.png" style="width:300px; height:250px;" alt="u8">
+
  <tr align="center">
−
</td>
+
    <td>1</td><td>94</td><td>120</td>
−
<td style="border-style: none;">
+
  </tr>
−
<img src="https://static.igem.org/mediawiki/2016/3/39/T--HokkaidoU_Japan--u9.png" style="width:300px; height:250px;" alt="u9">
+
  <tr align="center">
−
</td>
+
    <td>2</td><td>98</td><td>10</td>
−
</tr>
+
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>74</td><td>30 / 1kbp</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>4</td><td>98</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>5</td><td>72</td><td>30</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>6</td><td>98</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>7</td><td>70</td><td>30</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>8</td><td>98</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>9</td><td>68</td><td>30</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>10</td><td>68</td><td>420</td>
 +
  </tr>
 +
  <tr align="center">
 +
<td>11</td><td>4</td><td>Hold</td>
 +
  </tr>
  
−
</center>
 
 
</table>
 
</table>
−
<br clear="all">
+
Cycle:
 +
<br>sequence 2~3 &times; 5
 +
<br>sequence 4~5 &times; 5
 +
<br>sequence 6~7 &times; 5
 +
<br>sequence 8~9 &times; 15
  
−
<br>
+
</span>
−
<img src="https://static.igem.org/mediawiki/2016/f/f2/T--HokkaidoU_Japan--Universal_Festival_ZU33.jpg" style="width:800px; height:444px; " alt="Universal_Festival">
+
</div>
 +
<script>
 +
document.getElementById("disp1").style.display="none";
 +
var displaying=0;
 +
function hyoji1()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp1").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp1").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<span class="nomal2">
 
−
<h2>Comments</h2>
 
−
<p>Participant1</p>
 
  
−
<p>Age: 10’s / Sex: M</p>
 
−
<p>Score before workshop:5</p>
 
−
<p>Because genetically modified food is a little scary.</p>
 
  
−
<p>Score after workshop:8</p>
 
−
<p>Because I don't care if it's safe.</p>
 
−
<br />
 
  
−
<p>Participant2</p>
+
<h1 onClick="hyoji2()"><span class="ka-soru">PCR Purification</span></h1>
  
−
<p>Age: 20’s / Sex: F</p>
+
<div id="disp2">
−
<p>Score before workshop:0</p>
+
<span class="nomal2">
−
<p>Because it may be bad for the health.</p>
+
FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
 +
<br>Purification of PCR products
 +
</span>
  
−
<p>Score after workshop:6</p>
+
</div>
−
<p>Because it's safe to eat.</p>
+
<script>
 +
document.getElementById("disp2").style.display="none";
 +
var displaying=0;
 +
function hyoji2()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp2").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp2").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<br />
+
<h1 onClick="hyoji3()"><span class="ka-soru">Digestion</span></h1>
−
<p>Participant3
+
<div id="disp3">
 +
<span class="nomal2">
 +
Mix the following reagents in PCR tube.
 +
<table>
 +
  <tr align="center">
 +
    <th>Solution</th>
 +
    <td>DNA</td>
 +
    <td>RE1 10 U/&micro;L</td>
 +
    <td>RE2 10 U/&micro;L</td>
 +
    <td>Appropriate buffer</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>16</td>
 +
    <td>1</td>
 +
    <td>1</td>
 +
    <td>2</td>
 +
    <td>20</td>
 +
  </tr>
 +
</table>
 +
<table>
 +
  <tr align="center">
 +
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>1</td><td>37</td><td>120</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>2</td><td>65</td><td>15</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
</span>
 +
</div>
  
−
<p>Age: 40’s / Sex: F</p>
+
<script>
−
<p>Score before workshop:3</p>
+
document.getElementById("disp3").style.display="none";
−
<p>Because I suspect genetically modified foods or crops, harmful to noxious insects, is also harmful to human beings.</p>
+
var displaying=0;
 +
function hyoji3()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp3").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp3").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<p>Score after workshop:N/A</p>
 
−
<p>Because I can't answer since I didin't have time to see your poster.</p>
 
  
−
<br />
+
<h1 onClick="hyoji4()"><span class="ka-soru">Ligation</span></h1>
−
<p>Participant4</p>
+
<div id="disp4">
−
 
+
<span class="nomal2">
−
<p>Age: 10’s / Sex: M</p>
+
Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer.
−
<p>Score before workshop:3</p>
+
−
<p>I thought human being should not enter the territory of the gene.</p>
+
−
 
+
−
<p>Score after workshop:6</p>
+
−
<p>I thought whether it has bad effects on the environment depends on how we use it, and we can make our life more convenient without the effect.</p>
+
−
 
+
−
<br />
+
−
<p>Participant5</p>
+
−
 
+
−
<p>Age: 10’s / Sex: F</p>
+
−
<p>Score before workshop:4</p>
+
−
<p>Because it will have effect on the ecosystem.</p>
+
−
 
+
−
<p>Score after workshop:7</p>
+
−
<p>Because I understood that it can give various functions useful for human being.</p>
+
−
 
+
−
<br />
+
−
<p>Participant6</p>
+
−
 
+
−
<p>Age: 20’s / Sex: F</p>
+
−
<p>Score before workshop:3</p>
+
−
<p>Because it may be bad for the health.</p>
+
−
 
+
−
<p>Score after workshop:6
+
−
<p>Though it may be unhealthy to eat, I feel that it is acceptable.</p>
+
−
 
+
−
<br />
+
−
<p>Participant7</p>
+
−
 
+
−
<p>Age: 10’s / Sex: F</p>
+
−
<p>Score before workshop:6</p>
+
−
<p>Because there're indications whether the food such as soybeans use genetic modification.</p>
+
−
 
+
−
<p>Score after workshop:8</p>
+
−
<p>Because it was really fun to see real DNA.</p>
+
−
 
+
−
<br />
+
−
<p>Participant8</p>
+
−
 
+
−
<p>Age: 20’s / Sex: F</p>
+
−
<p>Score before workshop:5</p>
+
−
<p>It's very useful if we use it in a right way but it'll cause problems if we don't.</p>
+
−
 
+
−
<p>Score after workshop:5</p>
+
−
<p>The same reason.</p>
+
−
 
+
−
<br />
+
−
<p>Participant9</p>
+
−
 
+
−
<p>Age: 30’s / Sex: F</p>
+
−
<p>Score before workshop:5</p>
+
−
<p>I can't say good or bad.</p>
+
−
 
+
−
<p>Score after workshop:5</p>
+
−
<p>My child is in hospital because of a genopathy. I hope more and more remedies are dveloped to save lives.</p>
+
  
 +
<table>
 +
  <tr align="center">
 +
    <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td>
 +
  </tr>
 +
</table>
 +
Thermal protocol is following
 +
<table>
 +
  <tr align="center">
 +
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>1</td><td>16</td><td>30</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>2</td><td>65</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 
</span>
 
</span>
−
 
 
</div>
 
</div>
−
<br>
+
<script>
−
<div>
+
document.getElementById("disp4").style.display="none";
−
<img src="https://static.igem.org/mediawiki/2016/5/5b/T--HokkaidoU_Japan--Open_campus.png"  
+
var displaying=0;
−
width="270px" height="90px" alt="Open_campus">
+
function hyoji4()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp4").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp4").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
 +
<h1 onClick="hyoji5()"><span class="ka-soru">Electrophoresis</span></h1>
 +
<div id="disp5">
 
<span class="nomal2">
 
<span class="nomal2">
−
<br>
+
<ol>
−
<table  style="border-style: none; float: right;" height="400px" width="400px">
+
  <li>Put gel into electrophoresis tank.</li>
−
<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/4/4a/T--HokkaidoU_Japan--HumanPractice_OpenCampas1.jpg"
+
  <li>Pour 2x TBE buffer into the tank to soak gel.</li>
−
width="300px" height="auto" alt="Open_campus"></center></td></tr>
+
  <li>Add 5  &micro;L  of EtBr into cathode.</li>
−
+
  <li>Pre-migration for 30 min at 100 V.</li>
−
<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/9/96/T--HokkaidoU_Japan--HumanPractice_OpenCampas2.jpg"
+
  <li>Apply DNA solutions with 6x loading dye and ladder.</li>
−
width="300px" height="auto" alt="Open_campus"></center></td></tr>
+
  <li>Start electrophoresis at 100 V.</li>
−
</table>
+
  <li>Stop at appropriate time.
−
 
+
</ol>
−
<br>On 7th August, university’s open day for mainly high school students (called “Open Campus” in Japan), we gave a lecture and took a questionnaire. In the lecture, we explained what gene recombination is and about this year’s our project. According to the questionnaire, we can see that majority had an adverse impression on gene recombination before the lecture, however, more than 90% of visitors appreciated its usefulness by the end. Also, more than 90% of the people answered they acquired new knowledge about gene, DNA, protein and gene recombination. Based on these statistics, we consider that we accomplished contributing to the public understanding about gene and its recombination – topics which still raise anxiety amongst many people in Japan probably due to ignorance. We believe that our efforts will help people create a society in the future that utilizes gene recombination.
+
 
</span>
 
</span>
−
<br clear ="all">
 
−
<br>
 
  
−
<table style="border-style: none">
+
</div>
−
+
<script>
−
<tr align="center" style="border-style: none">
+
document.getElementById("disp5").style.display="none";
−
<td style="border-style: none;"><span class="small">SEX</span></td>
+
var displaying=0;
−
<td style="border-style: none;"><span class="small">AGE</span></td>
+
function hyoji5()
−
<td style="border-style: none;"><span class="small">School</span></td>
+
{
−
</tr>
+
  if (displaying==0)
 +
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 +
    document.getElementById("disp5").style.display="block";
 +
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  }
 +
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 +
  {
 +
    document.getElementById("disp5").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<tr align="center" style="border-style: none">
 
−
<td style="border-style: none;">
 
−
<img src="https://static.igem.org/mediawiki/2016/b/ba/T--HokkaidoU_Japan--o1.png" style="width:300px; height:250px;" alt="o1">
 
−
</td>
 
−
<td style="border-style: none;">
 
−
<img src="https://static.igem.org/mediawiki/2016/1/1d/T--HokkaidoU_Japan--o2.png" style="width:300px; height:250px;" alt="o2">
 
−
</td>
 
−
<td style="border-style: none;">
 
−
<img src="https://static.igem.org/mediawiki/2016/2/26/T--HokkaidoU_Japan--o3.png" style="width:300px; height:250px;" alt="o3">
 
−
</td>
 
−
</tr>
 
−
 
−
</table>
 
−
<br clear="all">
 
  
 +
<h1 onClick="hyoji6()"><span class="ka-soru">Gel Extraction</span></h1>
 +
<div id="disp6">
 
<span class="nomal2">
 
<span class="nomal2">
−
 
+
FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
−
<br>On a scale of 1 (do not consider, low and bad) to 5 (heavily consider, high, good)
+
<br>DNA extraction from gel
  
 
</span>
 
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp6").style.display="none";
 +
var displaying=0;
 +
function hyoji6()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp6").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp6").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<table style="border-style: none">
 
  
−
<tr align="center" style="border-style: none">  
+
<h1 onClick="hyoji7()"><span class="ka-soru">Ethanol precipitation</span></h1>
−
<td style="border-style: none;"><span class="small">Did you know about the DNA,<br>gene,and protein?</span></td>
+
<div id="disp7">
−
<td style="border-style: none;"><span class="small">Did you understand what gene, DNA, proteins are after taking the lecture? Did you learn anything new?</span></td>
+
<span class="nomal2">
−
<td style="border-style: none;"><span class="small">Did you know about GMO before taking the lecture? If you knew, did you have any idea what GMO is used for?</span></td>
+
<ol>
−
</tr>
+
  <li>Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.</li>
 +
  <li>Leave it at room temperature for 5 min.</li>
 +
  <li>Centrifuge at 15,000 rpm for 15 min at 25&deg;C.</li>
 +
  <li>Remove supernatant and add  600 &micro;L  of 70% ethanol.</li>
 +
  <li>Centrifuge at 15,000 rpm for 5 min at 25&deg;C.</li>
 +
  <li>Remove supernatant and dry up at room temperature with light sheilding.</li>
 +
  <li>Suspend with 10  &micro;L  of TE.</li>
 +
</ol>
  
−
<tr align="center" style="border-style: none">  
+
</span>
−
<td style="border-style: none;">
+
</div>
−
<img src="https://static.igem.org/mediawiki/2016/2/21/T--HokkaidoU_Japan--o4.png" style="width:300px; height:250px;" alt="o4">
+
<script>
−
</td>
+
document.getElementById("disp7").style.display="none";
−
<td style="border-style: none;">
+
var displaying=0;
−
<img src="https://static.igem.org/mediawiki/2016/a/aa/T--HokkaidoU_Japan--o5.png" style="width:300px; height:250px;" alt="o5">
+
function hyoji7()
−
</td>
+
{
−
<td style="border-style: none;">
+
  if (displaying==0)
−
<img src="https://static.igem.org/mediawiki/2016/e/e4/T--HokkaidoU_Japan--o7.png" style="width:300px; height:250px;" alt="o6">
+
  {
−
</td>
+
    document.getElementById("disp7").style.display="block";
−
</tr>
+
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp7").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<tr align="center" style="border-style: none">
+
<h1 onClick="hyoji8()"><span class="ka-soru">Colony PCR</span></h1>
−
<td style="border-style: none;"><span class="small"><br>Did you get any new knowledge<br>about GMO?</span></td>
+
<div id="disp8">
−
<td style="border-style: none;"><span class="small"><br>Based on today's lecture, do you think GMO is a useful technology?</span></td>
+
<span class="nomal2">
−
</tr>
+
<table>
  
−
<tr align="center" style="border-style: none">
+
  <tr align="center">
−
<td style="border-style: none;">
+
−
<img src="https://static.igem.org/mediawiki/2016/2/22/T--HokkaidoU_Japan--o8.png" style="width:300px; height:250px;" alt="o7">
+
−
</td>
+
−
<td style="border-style: none;">
+
−
<img src="https://static.igem.org/mediawiki/2016/6/66/T--HokkaidoU_Japan--o9.png" style="width:300px; height:250px;" alt="o8">
+
−
</td>
+
−
</tr>
+
  
 +
    <th>Solution</th>
 +
    <td>DNA</td>
 +
    <td>Kapa-Taq (Taq polymerase)</td>
 +
    <td>EX-F primer 10 &micro;M</td>
 +
    <td>PS-R primer 10 &micro;M</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>4.2</td>
 +
    <td>5</td>
 +
    <td>0.4</td>
 +
    <td>0.4</td>
 +
    <td>10</td>
 +
  </tr>
 
</table>
 
</table>
−
<br clear="all">
 
  
−
<span class="nomal2">
+
<table>
−
 
+
  <tr align="center">
−
<br>On a scale of 1 (do not consider, low and bad) to 10 (heavily consider, high, good)
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>1</td><td>94</td><td>120</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>2</td><td>94</td><td>30</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>68</td><td>60 / 1kbp</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>4</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycles: sequence 2~3 &times; 25~45
  
 
</span>
 
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp8").style.display="none";
 +
var displaying=0;
 +
function hyoji8()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp8").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp8").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<table style="border-style: none">
+
<h1 onClick="hyoji9()"><span class="ka-soru">Sequencing</span></h1>
 +
<div id="disp9">
 +
<span class="nomal2">
 +
<table>
 +
  <tr align="center">
 +
    <th>Solution</th>
 +
    <td>5 x Sequencing Buffer</td>
 +
    <td>primer 1 &micro;M</td>
 +
    <td>template DNA</td>
 +
    <td>Ready Reaction Premix</td>
 +
    <td>DW</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>1.5</td>
 +
    <td>1.5</td>
 +
    <td>1</td>
 +
    <td>1</td>
 +
    <td>5</td>
 +
    <td>10</td>
 +
  </tr>
 +
</table>
  
−
 
+
<table>
−
<tr align="center" style="border-style: none">  
+
  <tr align="center">
−
<td style="border-style: none;"><span class="small">What was your image of GMO before taking the lecture?</span></td>
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
−
</tr>
+
  </tr>
−
 
+
  <tr align="center">
−
<tr align="center" style="border-style: none">  
+
    <td>1</td><td>96</td><td>10</td>
−
<td style="border-style: none;">
+
  </tr>
−
<img src="https://static.igem.org/mediawiki/2016/d/d6/T--HokkaidoU_Japan--o6.png" style="width:300px; height:250px;" alt="o7">
+
  <tr align="center">
−
</td>
+
    <td>2</td><td>50</td><td>5</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>60</td><td>240</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>4</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycle: sequence 2~4 &times; 25
  
  
 +
<h2>Ethanol precipitation</h2>
 +
<table>
 +
  <tr align="center">
 +
    <th>Solution</th>
 +
    <td>PCR product</td>
 +
    <td>DW</td>
 +
    <td>3M NaOAc</td>
 +
    <td>Glycogen</td>
 +
    <td>100% EtOH</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>10</td>
 +
    <td>10</td>
 +
    <td>2</td>
 +
    <td>1</td>
 +
    <td>50</td>
 +
  </tr>
 
</table>
 
</table>
−
<br clear="all">
+
<ol>
 +
  <li>Centrifuge at 15,000 rpm for 15 min at room temprature.</li>
 +
  <li>Remove supernatant, add 100 &micro;L  of 70% EtOH and tap tubes by finger.</li>
 +
  <li>Centrifuge at 15,000 rpm for 10 min at room temprature.</li>
 +
  <li>Remove supernatant and dry up at room temperature.</li>
 +
  <li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li>
 +
  <li>Set the plate and start electrophoresis.</li>
 +
</ol>
 +
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp9").style.display="none";
 +
var displaying=0;
 +
function hyoji9()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp9").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp9").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
  
 +
<h1 onClick="hyoji10()"><span class="ka-soru">Competent Cells</span></h1>
 +
<div id="disp10">
 +
<span class="nomal2">
 +
<ol>
 +
<li>Thaw original competent cells on ice.</li>
 +
<li>Add 5 &micro;L of original competent cells to 2 mL of LB.</li>
 +
<li>Incubate the cells for 16 hrs at 37&deg;C.</li>
 +
<li>Add 5 &micro;L, 50 &micro;L, and 500 &micro;L of original cells to 100 mL of LB.</li>
 +
<li>Incubate the cells at 130 rpm at 20&deg;C, until OD<sub>600</sub> reach 0.5.</li>
 +
<li>Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4&deg;C.</li>
 +
<li>Remove supernatant and add 75 mL of TB to each tube.</li>
 +
<li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4&deg;C.</li>
 +
<li>Remove supernatant and add 32 mL of TB.</li>
 +
<li>Add 32 &micro;L of DMSO 10 times.</li>
 +
<li>Take 50 &micro;L and freeze with liquid nitrogen.</li>
 +
</ol>
 +
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp10").style.display="none";
 +
var displaying=0;
 +
function hyoji10()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp10").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp10").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
 +
<h1 onClick="hyoji11()"><span class="ka-soru">Transformation</span></h1>
 +
<div id="disp11">
 +
<span class="nomal2">
 +
<ol>
 +
  <li>Add plasmid to thawed competent cells on ice.</li>
 +
  <li>Incubate on ice for 30 min.</li>
 +
  <li>Add to LB.</li>
 +
  <li>Incubate the cells for 2 hrs at 37&deg;C.</li>
 +
  <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
 +
  <li>Incubate the plate(s) at 37&deg;C for 16~20 hrs.</li>
 +
</ol>
 +
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp11").style.display="none";
 +
var displaying=0;
 +
function hyoji11()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp11").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp11").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
 +
<h1 onClick="hyoji12()"><span class="ka-soru">Mini-prep</span></h1>
 +
<div id="disp12">
 
<span class="nomal2">
 
<span class="nomal2">
 +
FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd)
 +
<br>fast / standard / low copy protocol
 +
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp12").style.display="none";
 +
var displaying=0;
 +
function hyoji12()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp12").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp12").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
 
+
<h1 onClick="hyoji13()"><span class="ka-soru">Streaking (Single colony isolation)</span></h1>
−
 
+
<div id="disp13">
−
<br>
+
<span class="nomal2">
−
<br>Beside today's lecture, do you have any idea for the application of GMO?</br>
+
<ol>
−
<ul>
+
  <li>Pick the colony with an inoculating loop from the agar plate．</li>
−
  <li>I have had negative impression on genetically modified food. But I learned they can take the place of limited resources. I don't have any ideas now (I'm sorry). I hope that you do your best.  </li>
+
  <li>Drag the loop across on a new agar plate.</li>
−
  <li>Creating flowers which have diverse colors</li>
+
  <li>Resterilize the loop and drag it across again.</li>
−
  <li>Recreating organisms that lived in the past</li>
+
  <li>Repeat method 3.</li>
−
  <li>Creating nutritious food</li>
+
</ol>
−
  <li>If genetic modification is applied to people who are born weak, they may be able to overcome diseases.</li>
+
−
  <li>Apply genetic modification to medicine</li>
+
−
  <li>The regenerative ability like the tail of a lizard (Perhaps it has already been realized).</li>
+
−
  <li>Producing a large quantity of good cocoons of silkworms</li>
+
−
  <li>Creating bacteria which makes the concentration of carbon dioxide of sea water lower / Evolution of human beings / Creating livestock which eat less food and water and grow fast</li>
+
−
  <li>People will use up oil someday. So I think creating material instead of plastic and using it by genetic modification are good ideas.</li>
+
−
  <li>If we can select or create the gene which resist cancer cells, we may be able to apply it to wide medical cures. </li>
+
−
</ul>
+
 
</span>
 
</span>
 
</div>
 
</div>
 +
<script>
 +
document.getElementById("disp13").style.display="none";
 +
var displaying=0;
 +
function hyoji13()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp13").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp13").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<br>
+
<h1 onClick="hyoji14()"><span class="ka-soru">PEG precipitation</span></h1>
−
<div>
+
<div id="disp14">
−
<img src="https://static.igem.org/mediawiki/2016/b/be/T--HokkaidoU_Japan--radio.png"  
+
<span class="nomal2">
−
width="220px" height="110px" alt="radio">
+
<ol>
 +
  <li>Add 13  &micro;L  of PEG to 20  &micro;L  of product(s).</li>
 +
  <li>Leave it at room temperature for 1 hr.</li>
 +
  <li>Centrifuge at 15,000 rpm for 20 min at 4&deg;C.</li>
 +
  <li>Remove supernatant and add  100 &micro;L  of 70% ethanol.</li>
 +
  <li>Centrifuge at 15,000 rpm for 2 min at 4&deg;C.</li>
 +
  <li>Remove supernatant and dry up at room temperature with light sheilding.</li>
 +
  <li>Suspend with 10  &micro;L  of TE.</li>
 +
</ol>
 +
</span>
 +
</div>
 +
<script>
 +
document.getElementById("disp14").style.display="none";
 +
var displaying=0;
 +
function hyoji14()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp14").style.display="block";
 +
displaying=1;
 +
  }
 +
  else
 +
  {
 +
    document.getElementById("disp14").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
 +
<h1 onClick="hyoji15()"><span class="ka-soru">Gel Free System</span></h1>
 +
<div id="disp15">
 
<span class="nomal2">
 
<span class="nomal2">
−
<br>
+
<h2>Preparation of biotinylated DNA fragments</h2>
 +
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.
  
−
<table style="border-style: none; float: right;" height="400px" width="400px">
+
<table>
−
<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/2/2f/T--HokkaidoU_Japan--HumanPractice_radio2.jpg"
+
  <tr align="center">
−
width="300px" height="auto" alt="radio"></center></td></tr>
+
    <th>Solution</th>
−
+
    <td>template DNA</td>
−
<tr><td style="border-style: none;"><center><img src="https://static.igem.org/mediawiki/2016/d/d3/T--HokkaidoU_Japan--HumanPractice_radio.jpg"
+
    <td>5'-biotinylated 100-UP primer 10 &micro;M</td>
−
width="300px" height="auto" alt="radio"></center></td></tr>
+
    <td>5'-biotinylated 200-DN primer 10 &micro;M</td>
 +
    <td>MgSO<sub>4</sub></td>
 +
    <td>dNTPs</td>
 +
    <td>10x Buffer</td>
 +
    <td>KOD Plus Neo</td>
 +
    <td>DW</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <th>Volume (&micro;L)</th>
 +
    <td>1</td>
 +
    <td>1.5</td>
 +
    <td>1.5</td>
 +
    <td>3</td>
 +
    <td>5</td>
 +
    <td>5</td>
 +
    <td>1</td>
 +
    <td>32</td>
 +
    <td>50</td>
 +
  </tr>
 
</table>
 
</table>
 +
Thermal protocol is following
 +
<h2>2STEP Cycle (Tm value &gt; 63&deg;C)</h2>
 +
<table>
 +
  <tr align="center">
 +
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>1</td><td>94</td><td>120</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>2</td><td>98</td><td>10</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>3</td><td>68</td><td>30 / 1kbp</td>
 +
  </tr>
 +
  <tr align="center">
 +
    <td>4</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycle: sequence 2~3 &times; (25~45)
  
−
<br>On 7th August, university’s open day for mainly high school students (called “Open Campus” in Japan), we gave a lecture and took a questionnaire. In the lecture, we explained what gene recombination is and about this year’s our project. According to the questionnaire, we can see that majority had an adverse impression on gene recombination before the lecture, however, more than 90% of visitors appreciated its usefulness by the end. Also, more than 90% of the people answered they acquired new knowledge about gene, DNA, protein and gene recombination. Based on these statistics, we consider that we accomplished contributing to the public understanding about gene and its recombination – topics which still raise anxiety amongst many people in Japan probably due to ignorance. We believe that our efforts will help people create a society in the future that utilizes gene recombination.
+
<h2>Preparation of magnetic beads</h2>
 +
<ol>
 +
  <li>Mix 2 &micro;L  of magnetic beads (SiMAG-Streptavidin) and 48 &micro;L  of TE by vibration using sonic-toothbrush.</li>
 +
  <li>Collect the beads by attracting them to one side in 0.2 mL  polypropylene tube using neodymium magnet.</li>
 +
  <li>Remove supernatant.</li>
 +
</ol>
  
 +
<h2>Fixation to magnetic beads</h2>
 +
<ol>
 +
  <li>Add 3 &micro;L  of PCR product (0.48 pmol) and 7 &micro;L  of TE to beads.</li>
 +
  <li>Mix by vibration using sonic-toothbrush.</li>
 +
  <li>Collect the beads using magnet.</li>
 +
  <li>Remove supernatant containing excess amount of free DNA fragment.</li>
 +
</ol>
  
−
<br>We broadcast a talk about synthetic biology and iGEM toward people in Japan and Indonesia on Radio PPI Jepang, from 20:00 to 20:30 on July 28th, 2016. We believe people gained knowledge about syn-bio and iGEM through our talk.  We spoke in English, Japanese and Indonesian on the radio. Also, Radio PPI Jepang is an online radio that anyone can listen to if they have access to the internet, so we could successfully tell people about syn-bio and iGEM, especially Japanese people who had heard about the radio from Yusuke, and Indonesian people listening to the radio all over the world. We talked about 4 things: what syn-bio is like; its history; its benefits; about iGEM and iGEM HokkaidoU team. First, we told listeners that DNA codes proteins, and that it is like a cooking recipe book. Second, we spoke about the history of syn-bio, from the finding of DNA double helix model to Human Genome Project. Third, we suggested many benefits of syn-bio, such as curing diseases or cleaning polluted environment. Finally, we introduced iGEM and iGEM HokkaidoU team, about when and where iGEM started, what iGEMers do, and our project in 2016.  In conclusion, we succeeded in broadcasting about syn-bio and iGEM, and then enlightening people, especially Japanese and Indonesian people, with knowledge about syn-bio. After we finish the Giant Jamboree in 2016, we will again broadcast what iGEM’s Giant Jamboree is like,
+
<h2>Double restriction digestion</h2>
−
and raise people’s interest in iGEM!
+
<ol>
 +
  <li>Add Digestion Premix containing 1 &micro;L  of 10x RE solution, 8 &micro;L  of DW and each 0.5 &micro;L of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li>
 +
  <li>Mix by pumping using pipette.</li>
 +
  <li>Incubate at 37 &deg;C for 30 min.</li>
 +
  <li>Collect the beads using magnet.</li>
 +
  <li>Obtain supernatant containing digested DNA fragment.</li>
 +
   <li>Purify the supernatant by ethanol precipitation.</li>
 +
</ol>
 
</span>
 
</span>
−
 
 
</div>
 
</div>
−
<br clear="all">
+
</div>
 
<br>
 
<br>
−
<div>
+
<br>
−
<img src="https://static.igem.org/mediawiki/2016/d/de/T--HokkaidoU_Japan--paper.png"  
+
<script>
−
width="220px" height="110px" alt="paper">
+
document.getElementById("disp15").style.display="none";
 +
var displaying=0;
 +
function hyoji15()
 +
{
 +
  if (displaying==0)
 +
  {
 +
    document.getElementById("disp15").style.display="block";
 +
displaying=1;
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  }
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  else
 +
  {
 +
    document.getElementById("disp15").style.display="none";
 +
displaying=0;
 +
  }
 +
}
 +
</script>
  
−
<span class="nomal2">
+
 
−
<br>We wrote an introduction about iGEM for school newsletter called “Gakunavi.”  The newsletter is for all the students at Hokkaido university, so we took care to make it easy to understand. We did not use difficult scientific words, and we compared iGEM with robot contest to make it easy for people to understand what iGEM is like. Also, we introduced about what Giant Jamboree is, and what our project this year is. Finally, we told students to search us “iGEM HokkaidoU.” People who read the newsletter said that they got interested in iGEM. We believe many students at Hokkaido university read it, got knowledge about iGEM, and got interested in iGEM!
+
 
−
</span>
+
</body>
  
 
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Revision as of 17:38, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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Protcols


PCR

Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution template DNA Primer-F 10 µM Primer-R 10 µM MgSO4 dNTPs 10x Buffer KOD Plus Neo DW Total
Volume (µL) 1 1 1 3 5 5 1 33 50
Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
36830 / 1kbp
44Hold
Cycle: sequence 2~3 × (25~45)

3STEP Cycle (Tm value < 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
3Tm30
46830 / 1kbp
54Hold
Cycle: sequence 2~4 × (25~45)

STEP DOWN

SequenceTemp. (°C)Time (sec)
194120
29810
37430 / 1kbp
49810
57230
69810
77030
89810
96830
1068420
114Hold
Cycle:
sequence 2~3 × 5
sequence 4~5 × 5
sequence 6~7 × 5
sequence 8~9 × 15

PCR Purification

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products

Digestion

Mix the following reagents in PCR tube.
Solution DNA RE1 10 U/µL RE2 10 U/µL Appropriate buffer Total
Volume (µL) 16 1 1 2 20
SequenceTemp. (°C)Time (min)
137120
26515
34Hold

Ligation

Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer.
SolutionVector DNAInsert DNADWMighty MixTotal
Volume (µL)122510
Thermal protocol is following
SequenceTemp. (°C)Time (min)
11630
26510
34Hold

Electrophoresis

  1. Put gel into electrophoresis tank.
  2. Pour 2x TBE buffer into the tank to soak gel.
  3. Add 5 µL of EtBr into cathode.
  4. Pre-migration for 30 min at 100 V.
  5. Apply DNA solutions with 6x loading dye and ladder.
  6. Start electrophoresis at 100 V.
  7. Stop at appropriate time.

Gel Extraction

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Ethanol precipitation

  1. Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
  2. Leave it at room temperature for 5 min.
  3. Centrifuge at 15,000 rpm for 15 min at 25°C.
  4. Remove supernatant and add 600 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 5 min at 25°C.
  6. Remove supernatant and dry up at room temperature with light sheilding.
  7. Suspend with 10 µL of TE.

Colony PCR

Solution DNA Kapa-Taq (Taq polymerase) EX-F primer 10 µM PS-R primer 10 µM Total
Volume (µL) 4.2 5 0.4 0.4 10
SequenceTemp. (°C)Time (sec)
194120
29430
36860 / 1kbp
44Hold
Cycles: sequence 2~3 × 25~45

Sequencing

Solution 5 x Sequencing Buffer primer 1 µM template DNA Ready Reaction Premix DW Total
Volume (µL) 1.5 1.5 1 1 5 10
SequenceTemp. (°C)Time (sec)
19610
2505
360240
44Hold
Cycle: sequence 2~4 × 25

Ethanol precipitation

Solution PCR product DW 3M NaOAc Glycogen 100% EtOH
Volume (µL) 10 10 2 1 50
  1. Centrifuge at 15,000 rpm for 15 min at room temprature.
  2. Remove supernatant, add 100 µL of 70% EtOH and tap tubes by finger.
  3. Centrifuge at 15,000 rpm for 10 min at room temprature.
  4. Remove supernatant and dry up at room temperature.
  5. Resuspend the pellet to HiDi formamide and remove to 96-well plate.
  6. Set the plate and start electrophoresis.

Competent Cells

  1. Thaw original competent cells on ice.
  2. Add 5 µL of original competent cells to 2 mL of LB.
  3. Incubate the cells for 16 hrs at 37°C.
  4. Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
  5. Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
  6. Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
  7. Remove supernatant and add 75 mL of TB to each tube.
  8. Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
  9. Remove supernatant and add 32 mL of TB.
  10. Add 32 µL of DMSO 10 times.
  11. Take 50 µL and freeze with liquid nitrogen.

Transformation

  1. Add plasmid to thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add to LB.
  4. Incubate the cells for 2 hrs at 37°C.
  5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
  6. Incubate the plate(s) at 37°C for 16~20 hrs.

Mini-prep

FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
fast / standard / low copy protocol

Streaking (Single colony isolation)

  1. Pick the colony with an inoculating loop from the agar plate．
  2. Drag the loop across on a new agar plate.
  3. Resterilize the loop and drag it across again.
  4. Repeat method 3.

PEG precipitation

  1. Add 13 µL of PEG to 20 µL of product(s).
  2. Leave it at room temperature for 1 hr.
  3. Centrifuge at 15,000 rpm for 20 min at 4°C.
  4. Remove supernatant and add 100 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 2 min at 4°C.
  6. Remove supernatant and dry up at room temperature with light sheilding.
  7. Suspend with 10 µL of TE.

Gel Free System

Preparation of biotinylated DNA fragments

Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution template DNA 5'-biotinylated 100-UP primer 10 µM 5'-biotinylated 200-DN primer 10 µM MgSO4 dNTPs 10x Buffer KOD Plus Neo DW Total
Volume (µL) 1 1.5 1.5 3 5 5 1 32 50
Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

SequenceTemp. (°C)Time (sec)
194120
29810
36830 / 1kbp
44Hold
Cycle: sequence 2~3 × (25~45)

Preparation of magnetic beads

  1. Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
  2. Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
  3. Remove supernatant.

Fixation to magnetic beads

  1. Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
  2. Mix by vibration using sonic-toothbrush.
  3. Collect the beads using magnet.
  4. Remove supernatant containing excess amount of free DNA fragment.

Double restriction digestion

  1. Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
  2. Mix by pumping using pipette.
  3. Incubate at 37 °C for 30 min.
  4. Collect the beads using magnet.
  5. Obtain supernatant containing digested DNA fragment.
  6. Purify the supernatant by ethanol precipitation.