Difference between revisions of "Team:Kyoto/MaterialsMethods"

 
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#jump{
 
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<body>
 
<body>
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  <div id="jump"><a href="#wrapper"><img src="https://static.igem.org/mediawiki/2017/c/c5/Kyoto_notebook_jump.png"></a></div>
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<div class="clear"></div>
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  <div id="BACKGROUND">
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<div id="jump">
−
  <div id="wrapper">
+
 
−
  <h1>Material&amp;Methods</h1>
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<a href="#wrapper">
 +
<img src="https://static.igem.org/mediawiki/2018/1/11/T--Kyoto--upbotton.jpg"></a></div>
 +
<div id="BACKGROUND">
 +
 
 +
 
 +
<div style='padding-top: 100px;'><h1 id="wrapper"><img src="https://static.igem.org/mediawiki/2018/d/df/T--Kyoto--materialmethod.png" width="30%"></div></h1>
 +
 
 
   <ul class="material">
 
   <ul class="material">
−
       <li><a href="#Parts">1) Parts</a></li>
+
       <li><a href="#Parts"><font face="Segoe UI" font color="#007081">1) Parts</font></a></li>
−
       <li><a href="#Primer">2) Primer list</a></li>
+
       <li><a href="#Primer"><font face="Segoe UI" font color="#007081">2) Primer list</font></a></li>
−
       <li><a href="#Materials">3) Materials</a></li>
+
       <li><a href="#Materials"><font face="Segoe UI" font color="#007081">3) Materials</font></a></li>
 
         <ul>
 
         <ul>
−
<li><a href="#Kit">3-1 Kit</a></li>
+
<li><a href="#Kit"><font face="Segoe UI" font color="#007081">3-1 Kit</font></a></li>
−
<li><a href="#Restriction Enzyme">3-2 Restriction Enzyme</a></li>
+
<li><a href="#Restriction Enzyme"><font face="Segoe UI" font color="#007081">3-2 Restriction Enzyme</font></a></li>
−
<li><a href="#Polymerase">3-3 Polymerase</a></li>
+
<li><a href="#Polymerase"><font face="Segoe UI" font color="#007081">3-3 Polymerase</font></a></li>
−
<li><a href="#DNA ligase">3-4 DNA ligase</a></li>
+
<li><a href="#DNA ligase"><font face="Segoe UI" font color="#007081">3-4 DNA ligase</font></a></li>
−
<li><a href="#Marker">3-5 Marker</a></li>
+
<li><a href="#Marker"><font face="Segoe UI" font color="#007081">3-5 Marker</font></a></li>
−
<li><a href="#Organism">3-6 Organism</a></li>
+
<li><a href="#Organism"><font face="Segoe UI" font color="#007081">3-6 Organism</font></a></li>
−
<li><a href="#Antibiotics">3-7 Antibiotics</a></li>
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<li><a href="#Antibiotics"><font face="Segoe UI" font color="#007081">3-7 Antibiotics</font></a></li>
−
<li><a href="#Equipment">3-8 Equipment</a></li>
+
<li><a href="#Equipment"><font face="Segoe UI" font color="#007081">3-8 Equipment</font></a></li>
−
<li><a href="#Backbone">3-9 Backbone</a></li>
+
<li><a href="#Backbone"><font face="Segoe UI" font color="#007081">3-9 Backbone</font></a></li>
−
<li><a href="#Buffer">3-10 Buffer</a></li>
+
<li><a href="#Buffer"><font face="Segoe UI" font color="#007081">3-10 Buffer</font></a></li>
−
<li><a href="#SDS-PAGE&Westernblotting">3-11 SDS-PAGE&Westernblotting</a></li>
+
<li><a href="#SDS-PAGE&Westernblotting"><font face="Segoe UI" font color="#007081">3-11 SDS-PAGE&Westernblotting</font></a></li>
 
<!--------------------------------------------------------- BLOCK Contents-2 Materials  END----------------------------------------------------------->
 
<!--------------------------------------------------------- BLOCK Contents-2 Materials  END----------------------------------------------------------->
−
         </ul>
+
         </ul>
−
 
+
   <li><a href="#methods"><font face="Segoe UI" font color="#007081">4) Methods</font></a></li>
−
   <li><a href="#methods">4) Methods</a></li>
+
 
     <ul>
 
     <ul>
  
 
<!--------------------------------------------------------- BLOCK Contents-3 Methods PY------------------------------------------------------------>
 
<!--------------------------------------------------------- BLOCK Contents-3 Methods PY------------------------------------------------------------>
−
<li><a href="#Miniprep">4-1 Miniprep</a></li>
+
<li><a href="#Miniprep"><font face="Segoe UI" font color="#007081">4-1 Miniprep</font></a></li>
−
<li><a href="#Gel Extraction and PCR purification">4-2 Gel Extraction and PCR purification</a></li>
+
<li><a href="#Gel Extraction and PCR purification"><font face="Segoe UI" font color="#007081">4-2 Gel Extraction and PCR purification</font></a></li>
−
<li><a href="#Restriction Enzyme Digestion">4-3 Restriction Enzyme Digestion</a></li>
+
<li><a href="#Restriction Enzyme Digestion"><font face="Segoe UI" font color="#007081">4-3 Restriction Enzyme Digestion</font></a></li>
−
<li><a href="#Ligation">4-4 Ligation</a></li>
+
<li><a href="#Ligation"><font face="Segoe UI" font color="#007081">4-4 Ligation</font></a></li>
−
<li><a href="#Transformation">4-5 Transformation</a></li>
+
<li><a href="#Transformation"><font face="Segoe UI" font color="#007081">4-5 Transformation</font></a></li>
−
<li><a href="#PCR">4-6 PCR</a></li>
+
<li><a href="#PCR"><font face="Segoe UI" font color="#007081">4-6 PCR</font></a></li>
−
<li><a href="#Sequencing">4-7 Sequencing</a></li>
+
<li><a href="#Sequencing"><font face="Segoe UI" font color="#007081">4-7 Sequencing</font></a></li>
−
<li><a href="#Western blotting">4-8 Western blotting</a></li>
+
<li><a href="#Western blotting"><font face="Segoe UI" font color="#007081">4-8 Western blotting</font></a></li>
−
<li><a href="#Overnight-Culture">4-9 Overnight-Culture</a></li>
+
<li><a href="#Overnight-Culture"><font face="Segoe UI" font color="#007081">4-9 Overnight-Culture</font></a></li>
−
<li><a href="#Plates">4-10 Plates</a></li>
+
<li><a href="#Plates"><font face="Segoe UI" font color="#007081">4-10 Plates</font></a></li>
 
<!--------------------------------------------------------- BLOCK Contents-3 Methods  END------------------------------------------------------------>
 
<!--------------------------------------------------------- BLOCK Contents-3 Methods  END------------------------------------------------------------>
 
   </ul>
 
   </ul>
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<!-- ######################################################  BLOCK Contents END #################################################-->
 
<!-- ######################################################  BLOCK Contents END #################################################-->
  
 +
<br><br><br>
  
  
 
<!-----------------------------------------------------BLOCK Parts-------------------------------------------------------------->
 
<!-----------------------------------------------------BLOCK Parts-------------------------------------------------------------->
−
<h5 id="Parts">1) Parts</h5>
+
<h5 id="Parts">1)Parts</h5>
−
<h6><a href="https://2017.igem.org/Team:Kyoto/Basic_Part" target="brank">Basic Parts</a></h6>
+
 
−
<h6><a href="https://2017.igem.org/Team:Kyoto/Composite_Part" target="brank">Composite Parts</a></h6>
+
<h6><a href="https://2018.igem.org/Team:Kyoto/Basic_Parts"><font face="Segoe UI" font color="#007081">Basic Parts</font></a></h6>
 +
<h6><a href="https://2018.igem.org/Team:Kyoto/Composite_Parts"><font face="Segoe UI" font color="#007081">Composite Parts</font></a></h6><br><br><br>
 +
 
 
<!-----------------------------------------------------BLOCK Parts END---------------------------------------------------------->
 
<!-----------------------------------------------------BLOCK Parts END---------------------------------------------------------->
  
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<!------------------------------------------------------BLOCK primer PY----------------------------------------------------------><!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!------------------------------------------------------BLOCK primer PY----------------------------------------------------------><!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h5 id = "Primer">2) Primer List</h5>
+
<h5 id="Primer list">2)Primer_list</h5>
 +
 
 
<div class="example">
 
<div class="example">
 
<table width="20px">
 
<table width="20px">
−
<tr><th>primer name</th><th class="example" width=40%>sequence</th><th width=8%>Length</th><th width=8%>GC%</th><th width=5%>Tm</th><th width=10%>Designer</th><th>Manufacturer</th></tr>
+
<tr><th>primer name</th><th class="example" width=40%>sequence</th><th width=8%>Length</th><th width=7%>GC%</th><th width=5%>Tm</th><th width=10%>Designer</th><th width=14%>Manufacturer</th></tr>
 +
<tr><td>Z001</td><td>GTTAGGGCAGGGATGTAGATT</td><td>21</td><td>47.62</td><td>53.07</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z002</td><td>TGGTTAACGTATTCTCGATGTAAAG</td><td>25</td><td>36</td><td>53.19</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z003</td><td>TGGTTACAAACTACCTACAATTTG</td><td>24</td><td>33.33</td><td>51.29</td><td> Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z004</td><td>GATCTTTTACCTGATTTCGACC</td><td>22</td><td>40.91</td><td>51.18</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z005</td><td>TGTTCACACTTAATTCACATTTATTTGAGGCAACAATACGTGGCCAGCGACATGGAGGCCCAGAA</td><td>65</td><td>44.62</td><td>72.86</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z006</td><td>ATGAATAAGGAAAAAGATAGGGAGCACTTAATAGGCCCTGCCCTCGTTTAAACTGGATGGCGG</td><td>63</td><td>46.03</td><td>71.92</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z007</td><td>acacgctttttcagttcgagtttat</td><td>25</td><td>36</td><td>55.89</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z008</td><td>gtttcgaataaacacacataaacaaacaaaatgcgtaaaggagaagaacttttcactgga</td><td>60</td><td>33.33</td><td>66.8</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z009</td><td>tccagtgaaaagttcttctcctttacgcattttgtttgtttatgtgtgtttattcgaaac</td><td>60</td><td>33.33</td><td>66.8</td><td>Shimazoe</td><td>eurofin
 +
</td></tr>
 +
<tr><td>Z010</td><td>catggcatggatgaactatacaaataataaAACAGGTGGATCCCACATTGtcatgtaatt</td><td>60</td><td>35</td><td>66.71</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z011</td><td>aattacatgaCAATGTGGGATCCACCTGTTttattatttgtatagttcatccatgccatg</td><td>60</td><td>35</td><td>66.71</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z012</td><td>ggccgcaaattaaagccttcgagcg</td><td>25</td><td>56</td><td>63.35</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z013</td><td>gtttcgaataaacacacataaacaaacaaaatggcttcctccgaagacgttatcaaagag</td><td>60</td><td>36.67</td><td>67.57</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z014</td><td>ctctttgataacgtcttcggaggaagccattttgtttgtttatgtgtgtttattcgaaac</td><td>60</td><td>36.67</td><td>67.57</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z015</td><td>aataacgctgatagtgctagtgtagatcgcAACAGGTGGATCCCACATTGtcatgtaatt</td><td>60</td><td>41.67</td><td>69.94</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z016</td><td>aattacatgaCAATGTGGGATCCACCTGTTgcgatctacactagcactatcagcgttatt</td><td>60</td><td>41.67</td><td>69.94</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z017</td><td>gaattcgcggccgcttctagagaca</td><td>25</td><td>56</td><td>62.89</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z018</td><td>tgccggactgcagcggccgctacta</td><td>25</td><td>68</td><td>69.57</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z019</td><td>AAACATACTATTTAGGCTTGTTTATGTTCAGAACCTGTGACTCGTTTAAACTGGATGGCGGCGTT</td><td>65</td><td>40</td><td>70.47</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z020</td><td>atggccgctactgacagattaaacc</td><td>25</td><td>48</td><td>58.98</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z021</td><td>gagacccatcttgtaactcaatacg</td><td>25</td><td>44</td><td>55.16</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z022</td><td>gaataaacacacataaacaaacaaaatggccgctactgacagattaaacc</td><td>50</td><td>36</td><td>65.41</td><td> Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z023</td><td>ggtttaatctgtcagtagcggccattttgtttgtttatgtgtgtttattc</td><td>50</td><td>36</td><td>65.41</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z024</td><td>cgtattgagttacaagatgggtctcCACCACCACCACCATCACtagAACAGGTGGATCCCACATTGtcatg</td><td>71</td><td>49.3</td><td>73.64</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z025</td><td>catgaCAATGTGGGATCCACCTGTTctaGTGATGGTGGTGGTGGTGgagacccatcttgtaactcaatacg</td><td>71</td><td>49.3</td><td>73.64</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z026</td><td>gaattcgcggccgcttctagagacacgctttttcagttcgagtttat</td><td>47</td><td>46.81</td><td>69.83</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z027</td><td>tgccggactgcagcggccgctactagtaggccgcaaattaaagccttcgagcg</td><td>53</td><td>60.38</td><td>77.31</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z028</td><td>TGAAAACTCATTACCTAAATTTGTTTATGTTCGGTAGCCCCTCGTTTAAACTGGATGGCGGCGTT</td><td>65</td><td>41.54</td><td>71.34</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z029</td><td>GTATCCTTTTCAAGTACTTCCACCACCACCACCATCACta</td><td>40</td><td>45</td><td>65.52</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z030</td><td>taGTGATGGTGGTGGTGGTGGAAGTACTTGAAAAGGATAC</td><td>40</td><td>45</td><td>65.52</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z031</td><td>caacctcaatggagtgatgcaacc</td><td>24</td><td>50</td><td>58.35</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z032</td><td>AACAGGTGGATCCCACATTGtc</td><td>22</td><td>50</td><td>56.65</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z033</td><td>CCTTTGCTCTGACCGATCCATA</td><td>22</td><td>50</td><td>56.03</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z034</td><td>ACCCAGCACCATCAGAATTTAGCG</td><td>24</td><td>50</td><td>59.41</td><td>Shimazoe</td><td> eurofin</td></tr>
 +
<tr><td>Z035</td><td>CGGTGATTATCTAATCGAGGAAGAGG</td><td>26</td><td>46.15</td><td>56.42</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z036</td><td>GCTCATCAGTTCAATGGGAATCTTG</td><td>25</td><td>44</td><td>56.33</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z037</td><td>CAGCTTTGCTGCTATGTATGGTG</td><td>23</td><td>47.83</td><td>56.35</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z038</td><td>GGAACCGCAAAACCAGACTAC</td><td>21</td><td>52.38</td><td>55.81</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z039</td><td>tttgtttgtttatgtgtgtttattcgaaac</td><td>30</td><td>26.67</td><td>54.96</td><td>Shimazoe</td><td> eurofin</td></tr>
 +
<tr><td>Z040</td><td>AACAGGTGGATCCCACATTGtcatgtaatt</td><td>30</td><td>40</td><td>60.27</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z041</td><td>gtttcgaataaacacacataaacaaacaaa</td><td>30</td><td>26.67</td><td>54.96</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z042</td><td>aattacatgaCAATGTGGGATCCACCTGTT</td><td>30</td><td>40</td><td>60.27</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
<tr><td>Z043</td><td>GAAATTGCACTACCACCGGCGGCAAAATAT</td><td>30</td><td>46.67</td><td>63.65</td><td>Shimazoe</td><td>eurofin</td></tr>
 +
 
 +
<tr><td>Z044
 +
</td><td>cctatgaactgatggttggtg
 +
</td><td>21
 +
</td><td>47.62
 +
</td><td>52.62
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z045
 +
</td><td>ATACTGATGCTTCTGTAGAGGGTGA
 +
</td><td>25
 +
</td><td>44.00
 +
</td><td>56.62
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z046
 +
</td><td>GATAGCTTGGAGTTCATCGCGAGT
 +
</td><td>24
 +
</td><td>50.00
 +
</td><td>58.77
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z047
 +
</td><td>ggaagaggaggaagtgacatcgg
 +
</td><td>23
 +
</td><td>56.52
 +
</td><td>58.33
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z048
 +
</td><td>ggcCAAACATCCTCACCCTCTAC
 +
</td><td>23
 +
</td><td>56.52
 +
</td><td>58.81
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z049
 +
</td><td>ggacagttccatcgaggatc
 +
</td><td>20
 +
</td><td>55.00
 +
</td><td>53.93
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z050
 +
</td><td>agcttcttttcttctgcgcc
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>55.58
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z051
 +
</td><td>caggacgatggagtccaatg
 +
</td><td>20
 +
</td><td>55.00
 +
</td><td>54.50
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z052
 +
</td><td>atatcggcaccttcgacctg
 +
</td><td>20
 +
</td><td>55.00
 +
</td><td>56.03
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z053
 +
</td><td>GATGGACATAGGATCCTTAG
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>48.06
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z054
 +
</td><td>GCCACATTGTCTTTTGTTGC
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>53.22
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z055
 +
</td><td>CAGGACAGCGAGGCCGACTT
 +
</td><td>20
 +
</td><td>65.00
 +
</td><td>61.01
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z056
 +
</td><td>GCAGTTGCCACTGGTTCCGA
 +
</td><td>20
 +
</td><td>60.00
 +
</td><td>59.69
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z057
 +
</td><td>cgtatcgttgctcaaatgaa
 +
</td><td>20
 +
</td><td>40.00
 +
</td><td>51.00
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z058
 +
</td><td>tcaccacctgttccggtggt
 +
</td><td>20
 +
</td><td>60.00
 +
</td><td>59.82
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z059
 +
</td><td>GTAGAGTACAAGACGGCACT
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>53.16
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z060
 +
</td><td>CAGCGTCCTACGATAATACG
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>52.27
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z061
 +
</td><td>CCGGTTCTTCGTCCACTAAA
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>54.01
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z062
 +
</td><td>CTATTTCCAGTGGAGAAACG
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>50.25
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z063
 +
</td><td>ACAACAACCAGTTCGGAATC
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>52.73
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z064
 +
</td><td>TAGATCCCACCACCAACACGGCC
 +
</td><td>23
 +
</td><td>60.87
 +
</td><td>62.47
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z065
 +
</td><td>AGGCTATTACTGGTTATGGTCTTGG
 +
</td><td>25
 +
</td><td>44.00
 +
</td><td>55.94
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z066
 +
</td><td>agttgataatttcatcttaatagagc
 +
</td><td>26
 +
</td><td>26.92
 +
</td><td>49.61
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z067
 +
</td><td>acttcctcctcttccatc
 +
</td><td>18
 +
</td><td>50.00
 +
</td><td>49.02
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z068
 +
</td><td>GGATGATTTGCGTTATTGTTCTTAT
 +
</td><td>25
 +
</td><td>32.00
 +
</td><td>52.46
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z069
 +
</td><td>TTTCTCAAGCATGGGAAACAAGAGA
 +
</td><td>25
 +
</td><td>40.00
 +
</td><td>56.71
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z070
 +
</td><td>GCCATGGAAGACCCACCAAGA
 +
</td><td>21
 +
</td><td>57.14
 +
</td><td>58.53
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z071
 +
</td><td>ACGGCCAAAGAGAGCCATGG
 +
</td><td>20
 +
</td><td>60.00
 +
</td><td>59.19
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z072
 +
</td><td>GTCTTGGTGGGTCTTCCATG
 +
</td><td>20
 +
</td><td>55.00
 +
</td><td>54.58
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z073
 +
</td><td>GCGTTTCGAGGTCTTTGTAGCT
 +
</td><td>22
 +
</td><td>50.00
 +
</td><td>57.40
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z074
 +
</td><td>TCTACAGAAGCATCAGTATC
 +
</td><td>20
 +
</td><td>40.00
 +
</td><td>48.01
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z075
 +
</td><td>tcaatgtggttggttcagtgac
 +
</td><td>22
 +
</td><td>45.45
 +
</td><td>55.22
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z076
 +
</td><td>agatccatcccttctcgatgtc
 +
</td><td>22
 +
</td><td>50.00
 +
</td><td>55.22
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z077
 +
</td><td>GATGAAGTCGCTCTAGGTTGGG
 +
</td><td>22
 +
</td><td>54.55
 +
</td><td>56.66
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z078
 +
</td><td>ACACACGAATTCGCGGCCGCTTCTAGATGGAGCAAGGTAGTAATGTGAATCACCTG
 +
</td><td>56
 +
</td><td>50.00
 +
</td><td>72.76
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z079
 +
</td><td>CCCCACCTGCAGCGGCCGCTACTAGTATTATTATGTTTTTTCCGGTGGTAAATCTCCCTG
 +
</td><td>60
 +
</td><td>50.00
 +
</td><td>72.70
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z080
 +
</td><td>CACACAGAATTCGCGGCCGCTTCTAGATGAATGAAGAGGGCTTTTTTTTCAGTGCCAGAGGCCACCGTCCT
 +
</td><td>71
 +
</td><td>52.11
 +
</td><td>76.39
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z081
 +
</td><td>AAAATCACTGCAGCGGCCGCTACTAGTATTATTAGTCCAGAGGACGGTGGCCTCTGGCACTG
 +
</td><td>62
 +
</td><td>53.23
 +
</td><td>74.83
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z082
 +
</td><td>GAAAAAGAATTCGCGGCCGCTTCTAGATGCAAACATCCTCACCCTCTACAGAAGC
 +
</td><td>55
 +
</td><td>49.09
 +
</td><td>71.86
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z083
 +
</td><td>AGGGGGCTGCAGCGGCCGCTACTAGTATTATTACTTTGTACTAGGTGATGATGCAACAC
 +
</td><td>59
 +
</td><td>49.15
 +
</td><td>72.50
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z084
 +
</td><td>GAACTCCAAGCTATCTGATC
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>49.40
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z085
 +
</td><td>TGCCGACAGGAGAACagttgataatttcatcttaatagagc
 +
</td><td>41
 +
</td><td>39.02
 +
</td><td>63.55
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z086
 +
</td><td>GTTCTCCTGTCGGCAggtttagcctcgactacttt
 +
</td><td>35
 +
</td><td>51.43
 +
</td><td>66.58
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z087
 +
</td><td>TGCTGAAGTTGAATTggaaaattgggccaaagtag
 +
</td><td>35
 +
</td><td>40.00
 +
</td><td>63.03
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z088
 +
</td><td>AATTCAACTTCAGCAtcttccaccgatgtcacttc
 +
</td><td>35
 +
</td><td>42.86
 +
</td><td>63.62
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z089
 +
</td><td>GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGGGAACGAATGAAGAGGGCTTTTTTTTCAGTG
 +
</td><td>70
 +
</td><td>45.71
 +
</td><td>72.07
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z090
 +
</td><td>TTCTGTAGAGGGTGAGGATGTTTGTTAGGCTAGCTTACTCGCGATGA
 +
</td><td>47
 +
</td><td>46.81
 +
</td><td>69.00
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z091
 +
</td><td>GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGGGCACAGAGCAAGGTAGTAATG
 +
</td><td>61
 +
</td><td>49.18
 +
</td><td>71.68
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z092
 +
</td><td>CTGAAAAAGTACTTCTAGGCTCCCTTAGTGATGATGGTGATGGTGTCTGGA
 +
</td><td>51
 +
</td><td>45.10
 +
</td><td>68.47
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z093
 +
</td><td>ccGGCTAGCTTACTCGCGATGAA
 +
</td><td>23
 +
</td><td>56.52
 +
</td><td>60.84
 +
</td><td>Shimazoe
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z094
 +
</td><td>ATGCATGAATTCGCGGCCGCTTCTAGAGACACGCTTTTTCAGTTCGAGTT
 +
</td><td>50
 +
</td><td>48.00
 +
</td><td>72.04
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z095
 +
</td><td>TCCCCCCTGCAGCGGCCGCTACTAGTAGGCCGCAAATTA
 +
</td><td>39
 +
</td><td>61.54
 +
</td><td>74.13
 +
</td><td>Nambu
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z096
 +
</td><td>ATGGCTATCTGGGAGCAACT
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>54.79
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z097
 +
</td><td>ATCTACTGTCTGCAGAGTCAG
 +
</td><td>21
 +
</td><td>47.62
 +
</td><td>52.87
 +
</td><td>Tong
 +
</td><td>eurofin</td></tr><tr><td>Z098
 +
</td><td>TACGTAGGATCCCTGCAGTC
 +
</td><td>20
 +
</td><td>55.00
 +
</td><td>54.71
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z099
 +
</td><td>GACTAGCAGGAGGTGAAGAT
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>52.43
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z100
 +
</td><td>TGAAAGCGAAGACGAGATGG
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>54.49
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>Z101
 +
</td><td>AGAGCTTTAACAGCAGTAGG
 +
</td><td>20
 +
</td><td>45.00
 +
</td><td>51.22
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>VR
 +
</td><td>attaccgcctttgagtgagc
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>54.70
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>VF2
 +
</td><td>tgccacctgacgtctaagaa
 +
</td><td>20
 +
</td><td>50.00
 +
</td><td>55.05
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr><tr><td>Y001
 +
</td><td>ctaacggtacttctactgaag
 +
</td><td>21
 +
</td><td>42.86
 +
</td><td>49.31
 +
</td><td>Tong
 +
</td><td>eurofin
 +
</td></tr>
 +
 
 +
 
 +
 
 +
 
  
  
Line 128: Line 627:
  
 
</table>
 
</table>
−
</div>    <!-- Table end -->
+
</div>     
 +
<!-- Table end --><br><br><br>
 +
 
 
      
 
      
 
<!------------------------------------------------------BLOCK primer.py END---------------------------------------------------------->
 
<!------------------------------------------------------BLOCK primer.py END---------------------------------------------------------->
  
  
−
 
+
<h5 id="Materials">3)Materials</h5>
−
<h5 id="Materials">3) Materials</h5>
+
<!------------------------------------------------------BLOCK TableMaker PY------------------------------------------------------>
−
 
+
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<!------------------------------------------------------BLOCK TableMaker PY------------------------------------------------------><!-- Table Generated by TableMaker.py Author: OHAD -->
+
<h6 id = "Kit"><font face="Segoe UI">3-1 Kit</font></h6>
−
<h6 id = "Kit">3-1 Kit</h6>
+
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>Wizard&#174; SV Gel and PCR</td><td>Promega</td></tr><tr><td>FastGene™Plasmid Mini Kit</td><td>NIPPON Genetics Co.,Ltd</td></tr>
+
<tr><th width="300">Name</th><th width="300">Supplier</th></tr>
 +
<tr><td>FastGene™Plasmid Mini Kit</td><td>NIPPON Genetics Co., Ltd </td></tr>
 +
<tr><td>FastGene™Gel/PCR Extraction Kit </td><td>NIPPON Genetics Co.,Ltd</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Restriction Enzyme">3-2 Restriction Enzyme</h6>
+
<h6 id = "Restriction Enzyme"><font face="Segoe UI">3-2 Restriction Enzyme</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>EcoRI</td><td>TaKaRa, Promega</td></tr><tr><td>PstI</td><td>TaKaRa, Promega</td></tr><tr><td>SpeI</td><td>TaKaRa, Promega</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>EcoRI</td><td>TaKaRa, Promega</td></tr>
 +
<tr><td>PstI</td><td>TaKaRa, Promega</td></tr>
 +
<tr><td>SpeI</td><td>TaKaRa, Promega</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Polymerase">3-3 Polymerase</h6>
+
<h6 id = "Polymerase"><font face="Segoe UI">3-3 Polymerase</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>KAPA™HiFi HotStart ReadyMix (2x)</td><td>KAPABIOSYSTEMS</td></tr><tr><td>KAPA2G™ Fast HotStart ReadyMix with dye (2x)</td><td>KAPABIOSYSTEMS</td></tr><tr><td>KAPATaq™EXtra HotStart ReadyMix with dye</td><td>KAPABIOSYSTEMS</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>Q5 High-Fidelity 2x master mix</td><td>New England Biolabs Japan Inc.</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "DNA ligase">3-4 DNA ligase</h6>
+
<h6 id = "DNA ligase"><font face="Segoe UI">3-4 DNA ligase</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>T4 DNA ligase</td><td>TaKaRa</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>T4 DNA ligase</td><td>TaKaRa</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Marker">3-5 Marker</h6>
+
<h6 id = "Marker"><font face="Segoe UI">3-5 Marker</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>1kb DNA Ladder</td><td>TaKaRa</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>1kb DNA Ladder</td><td>NIPPON Genetics Co., Ltd</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Organism">3-6 Organism</h6>
+
<h6 id = "Organism"><font face="Segoe UI">3-6 Organism</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>E.coli DH5α Genotype</td><td>TaKaRa</td></tr><tr><td>E.coli BL21(DE3)pLysS Competent Cells</td><td>Promega</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>E.coli DH5α Genotype</td><td>TaKaRa</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Antibiotics">3-7 Antibiotics</h6>
+
<h6 id = "Antibiotics"><font face="Segoe UI">3-7 Antibiotics</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>Chloramphenicol</td><td>Wako</td></tr><tr><td>Ampicillin</td><td>Wako</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>Chloramphenicol</td><td>Wako</td></tr>
 +
<tr><td>Ampicillin</td><td>Wako</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Equipment">3-8 Equipment</h6>
+
<h6 id = "Equipment"><font face="Segoe UI">3-8 Equipment</font></h6>
 
<table>
 
<table>
−
<tr><th>Name </th><th>Supplier</th></tr><tr><td>BioPhotometer</td><td>eppendorf</td></tr><tr><td>LABO SHAKER</td><td>BIO CRAFT</td></tr><tr><td>CO2 INCUBATOR</td><td>SANYO</td></tr><tr><td>Pipette Controller</td><td>Biohit Midi Plus</td></tr><tr><td>MiniCentrifuge Model GMC-060</td><td>LMS CO.,LTD.</td></tr><tr><td>HIGH-SPEED REFRIGERATED CENTRIFUGE</td><td>TOMY</td></tr><tr><td>HIGH-PRESSURE STEAM STERILIZER</td><td>TOMY</td></tr><tr><td>VOTEX-GENE2</td><td>Scientific Industryes</td></tr><tr><td>Scanning Electron Miniscope TM1000s</td><td>HITACHI</td></tr><tr><td>Fluorescence Microscope BX61N-34-FL-1-D</td><td>OLYMPUS</td></tr><tr><td>SCIECE IMAGING SYSTEM LAS-3000</td><td>Fuji film</td></tr>
+
<tr><th>Name </th><th>Supplier</th></tr>
 +
<tr><td>BioPhotometer</td><td>eppendorf</td></tr>
 +
<tr><td>LABO SHAKER</td><td>BIO CRAFT</td></tr>
 +
<tr><td>CO2 INCUBATOR</td><td>SANYO</td></tr>
 +
<tr><td>Pipette Controller</td><td>Biohit Midi Plus</td></tr>
 +
<tr><td>MiniCentrifuge Model GMC-060</td><td>LMS CO.,LTD.</td></tr>
 +
<tr><td>HIGH-SPEED REFRIGERATED CENTRIFUGE</td><td>TOMY</td></tr>
 +
<tr><td>HIGH-PRESSURE STEAM STERILIZER</td><td>TOMY</td></tr>
 +
<tr><td>VOTEX-GENE2</td><td>Scientific Industryes</td></tr>
 +
<tr><td>Scanning Electron Miniscope TM1000s</td><td>HITACHI</td></tr>
 +
<tr><td>Fluorescence Microscope BX61N-34-FL-1-D</td><td>OLYMPUS</td></tr>
 +
<tr><td>SCIECE IMAGING SYSTEM LAS-3000</td><td>Fuji film</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 +
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Backbone">3-9 Backbone</h6>
+
<h6 id = "Backbone"><font face="Segoe UI">3-9 Backbone</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>pSB1C3</td><td>iGEM registry</td></tr><tr><td>pSB1A2</td><td>iGEM registry</td></tr><tr><td>pSB3C5</td><td>iGEM registry</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>pRS421</td><td>National BioResource Project<br>http://yeast.nig.ac.jp/yeast/</td></tr>
 +
<tr><td>pRS423 </td><td>National BioResource Project</td></tr>
 +
<tr><td>pRS424</td><td>National BioResource Project</td></tr>
 +
<tr><td>pRS425 </td><td>National BioResource Project</td></tr>
 +
<tr><td>pRS426</td><td>National BioResource Project</td></tr>
 +
<tr><td>pGK421</td><td>National BioResource Project</td></tr>
 +
<tr><td>pRS316 </td><td>National BioResource Project</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "Buffer">3-10 Buffer</h6>
+
<h6 id = "Buffer"><font face="Segoe UI">3-10 Buffer</font></h6>
 
<table>
 
<table>
−
<tr><th>Name</th><th>Supplier</th></tr><tr><td>Sodium Carbonate</td><td>Wako</td></tr><tr><td>Sodium Bicarbonate</td><td>Wako</td></tr><tr><td>Methanol(99.5%)</td><td>Wako</td></tr><tr><td>Sodium Chloride</td><td>Wako</td></tr><tr><td>SDS</td><td>nacalai tesque</td></tr><tr><td>Trisaminomethane</td><td>nacalai tesque</td></tr><tr><td>Potassium dihydrogenphosphste</td><td>SIGAMA-ALDRICH</td></tr><tr><td>Potassium Chloride</td><td>Wako</td></tr><tr><td>Glycine</td><td>Wako</td></tr><tr><td>Agar, Powder</td><td>Wako</td></tr><tr><td>Agarose XP</td><td>Wako</td></tr><tr><td>10% Hydrochloric Acid</td><td>Wako</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>Sodium Carbonate</td><td>Wako</td></tr>
 +
<tr><td>Sodium Bicarbonate</td><td>Wako</td></tr>
 +
<tr><td>Methanol(99.5%)</td><td>Wako</td></tr>
 +
<tr><td>Sodium Chloride</td><td>Wako</td></tr>
 +
<tr><td>SDS</td><td>nacalai tesque</td></tr>
 +
<tr><td>Trisaminomethane</td><td>nacalai tesque</td></tr>
 +
<tr><td>Potassium dihydrogenphosphste</td><td>SIGAMA-ALDRICH</td></tr>
 +
<tr><td>Potassium Chloride</td><td>Wako</td></tr>
 +
<tr><td>Glycine</td><td>Wako</td></tr>
 +
<tr><td>Agar, Powder</td><td>Wako</td></tr>
 +
<tr><td>Agarose XP</td><td>Wako</td></tr>
 +
<tr><td>10% Hydrochloric Acid</td><td>Wako</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
 
<!-- Table Generated by TableMaker.py Author: OHAD -->
−
<h6 id = "SDS-PAGE&Westernblotting">3-11 SDS-PAGE&Westernblotting</h6>
+
<h6 id = "SDS-PAGE&Westernblotting"><font face="Segoe UI">3-11 SDS-PAGE&Westernblotting</font></h6>
 
<table>
 
<table>
−
<tr><th>IMMOBILON - P Blotting Sandwiches</th><th>Immobilon</th></tr><tr><td>REAL GEL PLATE (10%)</td><td>BIO CRAFT</td></tr><tr><td>BE-210(SDS-PAGE)</td><td>BIO CRAFT</td></tr><tr><td>BE-330</td><td>BIO CRAFT</td></tr><tr><td>Amersham ECL Anti-Mouse IgG</td><td>GE healthcare</td></tr><tr><td>Amersham ECL Anti-rabbit IgG</td><td>GE healthcare</td></tr><tr><td>Amersham ECL Prime Blocking Reagent</td><td>GE healthcare</td></tr><tr><td>Amersham ECL Prime WB Detection Reagent</td><td>GE healthcare</td></tr>
+
<tr><th>Name</th><th>Supplier</th></tr>
 +
<tr><td>REAL GEL PLATE (10%)</td><td>BIO CRAFT</td></tr>
 +
<tr><td>BE-210(SDS-PAGE)</td><td>BIO CRAFT</td></tr>
 +
<tr><td>BE-330</td><td>BIO CRAFT</td></tr>
 +
<tr><td>Amersham ECL Anti-Mouse IgG</td><td>GE healthcare</td></tr>
 +
<tr><td>Amersham ECL Anti-rabbit IgG</td><td>GE healthcare</td></tr>
 +
<tr><td>Amersham ECL Prime Blocking Reagent</td><td>GE healthcare</td></tr>
 +
<tr><td>Amersham ECL Prime WB Detection Reagent</td><td>GE healthcare</td></tr>
 
</table>
 
</table>
 
<!-- Table end -->
 
<!-- Table end -->
Line 207: Line 756:
 
<tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr>
 
<tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr>
 
</table>
 
</table>
−
<!-- Table end -->
+
<!-- Table end --><br><br><br>
 +
 
  
 
<!------------------------------------------------------BLOCK TableMaker.py END------------------------------------------------------>
 
<!------------------------------------------------------BLOCK TableMaker.py END------------------------------------------------------>
  
  
−
<h5 id="methods">4) Methods</h5>
+
<h5 id="methods">4)Methods</h5>
 +
 
 
<!------------------------------------------------------BLOCK Methods PY---------------------------------------------------------------->
 
<!------------------------------------------------------BLOCK Methods PY---------------------------------------------------------------->
 
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−
<h6 id = "Miniprep">4-1 Miniprep</h6>
+
<h6 id = "Miniprep"><font face="Segoe UI">4-1 Miniprep</font></h6>
 
<p>
 
<p>
−
Minipreps were performed using FastGene&#8482;Plasmid Mini Kit and and Wizard® Plus SV Minipreps DNA Purification System according to the manufacturer's protocols.
+
Minipreps were performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
−
 
 
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<!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
−
<h6 id = "Gel Extraction and PCR purification">4-2 Gel Extraction and PCR purification</h6>
+
<h6 id = "Gel Extraction and PCR purification"><font face="Segoe UI">4-2 Gel Extraction and PCR purification</font></h6>
 
<p>
 
<p>
−
Gel Extraction was performed using Wizard® SV Gel and PCR Clean-Up System according to the manufacturer's protocols.  
+
Gel Extraction was performed using FastGene™Gel/PCR Extraction Kit according to the manufacturer's protocols.
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
−
 
 
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−
<h6 id = "Restriction Enzyme Digestion">4-3 Restriction Enzyme Digestion</h6>
+
<h6 id = "Restriction Enzyme Digestion"><font face="Segoe UI">4-3 Restriction Enzyme Digestion</font></h6>
 
<p>
 
<p>
−
Restriction enzyme treatment was performed using Takara Bio's or NEB's restriction enzymes according to the respective manufacturer's protocols.
+
Restriction enzyme treatment was performed using Q5 High-Fidelity 2x master mix according to the respective manufacturer's protocols.
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
−
 
 
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−
<h6 id = "Ligation">4-4 Ligation</h6>
+
<h6 id = "Ligation"><font face="Segoe UI">4-4 Ligation</font></h6>
 
<p>
 
<p>
−
Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's
+
Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's.
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
−
 
 
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−
<h6 id = "Transformation">4-5 Transformation</h6>
+
<h6 id = "Transformation"><font face="Segoe UI">4-5 Transformation</font></h6>
−
<ol>
+
<p>
−
<li>Thaw competent cells on ice</li>
+
1.Thaw competent cells on ice<br>
−
<li>Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes</li>
+
2.Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes</br>
−
<li>Keep them in heating block for 45 seconds, then cool on ice for 2 minutes</li>
+
3.Keep them in heating block for 45 seconds, then cool on ice for 2 minutes<br>
−
<li>Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37°C</li>
+
4.Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37℃<br>
−
<li>Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37°C</li>
+
5.Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37℃
−
</ol>
+
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
−
 
 
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−
<h6 id = "PCR">4-6 PCR</h6>
+
<h6 id = "PCR"><font face="Segoe UI">4-6 PCR</font></h6>
 
<p>
 
<p>
−
PCR was performed using KAPA™HiFi HotStart ReadyMix (2x), KAPA™ 2G Robust HotStart ReadyMix (2x), PrimeSTAR® Max DNA Polymerase, GoTaq®, Takara Ex Taq, and Quick Taq® HS DyeMix according to the manufacturer's protocols
+
PCR was performed using Q5 High-Fidelity 2x master mix according to the manufacturer's protocols.</p>
−
</p>
+
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 +
 +
  
 
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−
<h6 id = "Sequencing">4-7 Sequencing</h6>
+
<h6 id = "Sequencing"><font face="Segoe UI">4-7 Sequencing</font></h6>
 
<p>
 
<p>
 
We outsourced the sequencing to Macrogen
 
We outsourced the sequencing to Macrogen
Line 269: Line 816:
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 +
  
 
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−
<h6 id = "RT-PCR">4-8 RT-PCR</h6>
+
<h6 id = "Western blotting"><font face="Segoe UI">4-8 Western blotting</font></h6>
 
<p>
 
<p>
−
We extracted <I>B. xylophilus</I> whole mRNA using Sepazol®-RNA Ⅰ Super G according to the manufacturer's protocol.  
+
1.Apply sample to SDS-PAGE minigel (BIOCRAFT 15%).
 +
<br>2.Soak the gel in transfer buffer.
 +
<br>3.Soak PVDF membrane in 100% methanol for 30 sec.
 +
<br>4.Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min.
 +
<br>5.Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode.
 +
<br>6.Transfer the proteins from the gel to the membrane with 100 mA for 1 h.
 +
<br>7.Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h.
 +
<br>8.Wash for 5 min 3 times with TBST.
 +
<br>9.Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h.
 +
<br>10.Wash for 5 min 3 times with TBST.
 +
<br>11.Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h.
 +
<br>12.Wash for 5 min 3 times with TBST.
 +
<br>13.Drain excess wash buffer from the washed membrane and place on flat surface, protein side up.
 +
<br>14.Add detection reagent onto the membrane, covering all of the membrane.
 +
<br>15.Incubate for 5 minutes at room temperature.
 +
<br>16.Drain off excess detection reagent by dabbing with Kimwipe.
 +
<br>17.Place the sample in the CCD camera compartment and record the images.
 
</p>
 
</p>
 +
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 +
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 +
<h6 id = "Overnight-Culture"><font face="Segoe UI">4-9 Overnight-Culture</font> </h6>
 
<p>
 
<p>
−
RT-PCR was performed using ~~~ according to the manufacturer's protocol.
+
1.Prepare LB media by resolving 5g Bacto Tryptone, 1.25g Bacto-yeast extract and 1.25g NaCl in 500ml of milliQ and autoclave it.
 +
<br>2.Transfer 7ml of LB into a culture tube and add the respective antibiotic stock solutions.
 +
<br>3.Inoculate the culture by picking a single colony with a pipet tip and tipping it into the medium.
 +
<br>4.Shake the culture at 37℃ at 180 rpm for 14-16h.
 +
<br>5.Isolate plasmids or use the culture for further experiments.
 +
<table>
 +
<h7> Concentrations of antibiotics:</h7>
 +
<tr><th>Antibiotics</th><th>Stock concentration</th><th>Final concentration</th></tr>
 +
<tr><td>Ampicillin</td><td>100mg/ml</td><td>100μg/ml</td></tr>
 +
<tr><td>Chloramphenicol</td><td>10mg/l</td><td>30μg/ml</td></tr>
 +
</table>
 
</p>
 
</p>
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
  
 
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−
<h6 id = "RTqPCR">4-9 qRT-PCR</h6>
+
<h6 id = "Plates"><font face="Segoe UI">4-10 Plates</font></h6>
−
<p>We inserted 3 plasmids written on the lower left into 2 types of yeasts written on the lower right.
+
<p>
−
  <table class="pcr">
+
1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
−
      <tr>
+
<br>2. Transfer 1. to the flask.
−
      <td class="pcr">p14 Gal1p-AK1<br>
+
<br>3. Place the following in the flask of 2.
−
      p26 GPDp-AK1<br>
+
<br> ・Bacto Tryptone 5 g
−
      p100 GPF-GFP</td>
+
<br> ・Bacto yeast extract 2.5 g
−
      <td class="pcr">MKY13 WT<br>
+
<br> ・NaCl 2.5g
−
      MKY117 ski2Δ</td>
+
<br> ・Agar 7.5 g
−
      </tr>
+
<br>4. After mixing thoroughly, autoclave it after covering with aluminum foil.
−
  </table>
+
<br>5. Add the respective antibiotics.
−
Regarding p14, we conducted
+
<br>6. Cool until it gets moderately warm to the touch.
−
<ol>
+
<br>7. Put in a container and fix the lid with the tape after the medium solidifies.
−
<li>Create culture with raffinose medium as primary culture.</li>
+
</p>
−
<li>Dilute it in glucose medium and galactose medium and incubate for 12-15 hours.</li>
+
<table>
−
<li>Collect at log phase of OD = 1.1- 1.8 (MKY13) 0.6 - 1.2 .(MKY117) </li>
+
<h7> Concentrations of antibiotics:</h7>
−
<li>Collect RNA by Lucigen's MasterPure Yeast RNA purification kit. (http://www.lucigen.com/home.php?cat=273)</li>
+
<tr><th>Antibiotics</th><th>Stock concentration</th><th>Final concentration</th></tr>
−
<li>Do DNase treatment according to the manual for 20 minutes.</li>
+
<tr><td>Ampicillin</td><td>100mg/ml</td><td>100μg/ml</td></tr>
−
<li>Dissolve the obtained total RNA in 15 μL and dilute it to prepare a standard curve sample (using p14 sample and p100 sample). The measurement sample was diluted 5000 times with water and used. Analysis is ABI Step-one plus. SuperScript III Platinum SYBR qRT-PCR kit (https://www.thermofisher.com/order/catalog/product/11732020) was used.</li>
+
<tr><td>Chloramphenicol</td><td>10mg/ml</td><td>30μg/ml</td></tr>
−
</ol>
+
</table>
−
<p>The three following primers were used :
+
−
<ul class="material">
+
−
<li>IK91-IK92: This amplifies the loop region of hairpin RNA</li>
+
−
<li>IK95-IK96: This amplifies the inside of AK1 mRNA</li>
+
−
<li>Kota 030 - Kota 031: It amplifies near the 5 'end of 25S rRNA. (Fujii Kitabatake et al., 2009)</li>
+
−
</ul>
+
−
 
+
−
<p>Quantitation was performed with n = 3 biological triplicate, and the variation in RNA recovery amount was standardized by dividing the quantified value of loop and the quantified value of AK1 by the quantitative value of 25S rRNA, respectively.</p>
+
 
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
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−
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+
<br>
−
<h6 id = "Western blotting (Sample preparation of S. cerevisiae)">4-10 Western blotting (Sample preparation of <I>S. cerevisiae</I>)</h6>
+
<br>
−
<ol>
+
<br>
−
<li>Cultivate 1ml of  <I>S. cerevisiae</I> culture whose OD600 values are 1.0</li>
+
 
−
<li>Pour 1ml of the <I>S. cerevisiae</I> cell culture into 1.5ml tubes</li>
+
 
−
<li>Centrifuge 5000rpm for 1min and remove the supernatant</li>
+
 
−
<li>Resuspend with 30ul of SDS sample buffer </li>
+
 
−
<li>Heat samples for 10min at 100°C using block incubater</li>
+
 
−
<li>Vortex well</li>
+
 
−
</ol>
+
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
  
−
<!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
 
−
<h6 id = "Western blotting (Basic protocol)">4-11 Western blotting (Basic protocol)</h6>
 
−
<ol>
 
−
<li>Apply sample to SDS-PAGE minigel (BIOCRAFT 15%)</li>
 
−
<li>Soak the gel in transfer buffer</li>
 
−
<li>Soak PVDF membrane in 100% methanol for 30 sec</li>
 
−
<li>Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min</li>
 
−
<li>Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode</li>
 
−
<li>Transfer the proteins from the gel to the membrane with 100 mA for 1 h</li>
 
−
<li>Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h</li>
 
−
<li>Wash for 5 min 3 times with TBST</li>
 
−
<li>Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h</li>
 
−
<li>Wash for 5 min 3 times with TBST</li>
 
−
<li>Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h</li>
 
−
<li>Wash for 5 min 3 times with TBST</li>
 
−
<li>Drain excess wash buffer from the washed membrane and place on flat surface, protein side up</li>
 
−
<li>Add detection reagent onto the membrane, covering all of the membrane</li>
 
−
<li>Incubate for 5 minutes at room temperature</li>
 
−
<li>Drain off excess detection reagent by dabbing with Kimwipe</li>
 
−
<li>Place the sample in the CCD camera compartment and record the images</li>
 
−
</ol>
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
  
−
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−
<h6 id = "Culture using yeasts and measurement">4-12 Culture using yeasts and measurement</h6>
 
−
<ol>
 
−
<li>Prepare medium put on 3.5 cm plate.</li>
 
−
<li>Drop liquid-cultured yeasts on the center of medium.</li>
 
−
<li>Disperse them by bacteria spreader and dry for 30min to 1 hour. Or leave them as they are.</li>
 
−
<li>Pipette suspension including nematodes on the medium.</li>
 
−
<li>Culture it at 25 ℃</li>
 
−
</ol>
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
  
−
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−
<h6 id = "The way of taking photos">4-13 The way of taking photos</h6>
 
−
<ol>
 
−
<li>Culture yeasts expressing GFP over night and dilute them three times with DW or SD liquid medium.</li>
 
−
<li>Drop 200-300μl of water on the medium after one or two days, and pipette it in order to spread over the medium.</li>
 
−
<li>Suck out 12ul of water on the medium and drop it on microscope slide.</li>
 
−
<li>Cover it with cover glass and observe whether nematodes feed yeasts expressing GFP by fluorescence microscope.</li>
 
−
</ol>
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
  
−
<!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
 
−
<h6 id = "Methods of taking movies">4-14 Methods of taking movies</h6>
 
−
<ol>
 
−
<li>Make agar pad.</li>
 
−
<li>Extract nematodes from PDA medium, collect them into 1.5ml tube and centrifuge it.</li>
 
−
<li>Remove supernatant and put 1.5ul of suspension with nematodes into gap of cover glasses.</li>
 
−
<li>Snap microscope slide with the finger and reach suspension to the center of agarose medium.</li>
 
−
<li>Observe whether nematodes feed yeast expressing GFP by microscope and take videos of it.</li>
 
−
</ol>
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
  
−
<!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
 
−
<h6 id = "Way of making agar pad">4-15 Way of making agar pad</h2>
 
−
<ol>
 
−
<li>Make medium of 2% agar and sterlize by autoclave.</li>
 
−
<li>Put sterlized slide glass between two of slide glasses covered with "Kimwipes", drop agar medium, then rapidly cover agar medium with another slide glass.</li>
 
−
<li>After the medium sodifies, reveal slowly covered slide glass.</li>
 
−
<li>Pipette yeasts on the medium, cover the medium on cover glass, and culture it for 1-2days.  </li>
 
−
</ol>
 
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
−
<h6 id="dsRNA">4-16 Synthesis of dsRNA</h6>
 
−
<p>We synthesyzed dsRNA by "MEGAscript™ T7 Transcription Kit."<br><a href="https://www.thermofisher.com/order/catalog/product/AM1334">https://www.thermofisher.com/order/catalog/product/AM1334</a></p>
 
  
−
<!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
 
−
<h6 id = "Soaking">4-17 Soaking</h6>
 
−
<p>We followed this paper as for soaking.<br>
 
−
  <a href="https://link.springer.com/content/pdf/10.1007%2Fs10658-012-0035-0.pdf">https://link.springer.com/content/pdf/10.1007%2Fs10658-012-0035-0.pdf</a></p>
 
−
</ol>
 
−
<br>
 
−
<br>
 
  
  
Line 402: Line 908:
  
  
−
<!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
 
  
 
<!------------------------------------------------------BLOCK Methods.py END ----------------------------------------------------------->
 
<!------------------------------------------------------BLOCK Methods.py END ----------------------------------------------------------->

Latest revision as of 16:00, 17 October 2018




1)Parts
Basic Parts
Composite Parts



2)Primer_list
primer namesequenceLengthGC%TmDesignerManufacturer
Z001GTTAGGGCAGGGATGTAGATT2147.6253.07Shimazoeeurofin
Z002TGGTTAACGTATTCTCGATGTAAAG253653.19Shimazoeeurofin
Z003TGGTTACAAACTACCTACAATTTG2433.3351.29 Shimazoeeurofin
Z004GATCTTTTACCTGATTTCGACC2240.9151.18Shimazoeeurofin
Z005TGTTCACACTTAATTCACATTTATTTGAGGCAACAATACGTGGCCAGCGACATGGAGGCCCAGAA6544.6272.86Shimazoeeurofin
Z006ATGAATAAGGAAAAAGATAGGGAGCACTTAATAGGCCCTGCCCTCGTTTAAACTGGATGGCGG6346.0371.92Shimazoeeurofin
Z007acacgctttttcagttcgagtttat253655.89Shimazoeeurofin
Z008gtttcgaataaacacacataaacaaacaaaatgcgtaaaggagaagaacttttcactgga6033.3366.8Shimazoeeurofin
Z009tccagtgaaaagttcttctcctttacgcattttgtttgtttatgtgtgtttattcgaaac6033.3366.8Shimazoeeurofin
Z010catggcatggatgaactatacaaataataaAACAGGTGGATCCCACATTGtcatgtaatt603566.71Shimazoeeurofin
Z011aattacatgaCAATGTGGGATCCACCTGTTttattatttgtatagttcatccatgccatg603566.71Shimazoeeurofin
Z012ggccgcaaattaaagccttcgagcg255663.35Shimazoeeurofin
Z013gtttcgaataaacacacataaacaaacaaaatggcttcctccgaagacgttatcaaagag6036.6767.57Shimazoeeurofin
Z014ctctttgataacgtcttcggaggaagccattttgtttgtttatgtgtgtttattcgaaac6036.6767.57Shimazoeeurofin
Z015aataacgctgatagtgctagtgtagatcgcAACAGGTGGATCCCACATTGtcatgtaatt6041.6769.94Shimazoeeurofin
Z016aattacatgaCAATGTGGGATCCACCTGTTgcgatctacactagcactatcagcgttatt6041.6769.94Shimazoeeurofin
Z017gaattcgcggccgcttctagagaca255662.89Shimazoeeurofin
Z018tgccggactgcagcggccgctacta256869.57Shimazoeeurofin
Z019AAACATACTATTTAGGCTTGTTTATGTTCAGAACCTGTGACTCGTTTAAACTGGATGGCGGCGTT654070.47Shimazoeeurofin
Z020atggccgctactgacagattaaacc254858.98Shimazoeeurofin
Z021gagacccatcttgtaactcaatacg254455.16Shimazoeeurofin
Z022gaataaacacacataaacaaacaaaatggccgctactgacagattaaacc503665.41 Shimazoeeurofin
Z023ggtttaatctgtcagtagcggccattttgtttgtttatgtgtgtttattc503665.41Shimazoeeurofin
Z024cgtattgagttacaagatgggtctcCACCACCACCACCATCACtagAACAGGTGGATCCCACATTGtcatg7149.373.64Shimazoeeurofin
Z025catgaCAATGTGGGATCCACCTGTTctaGTGATGGTGGTGGTGGTGgagacccatcttgtaactcaatacg7149.373.64Shimazoeeurofin
Z026gaattcgcggccgcttctagagacacgctttttcagttcgagtttat4746.8169.83Shimazoeeurofin
Z027tgccggactgcagcggccgctactagtaggccgcaaattaaagccttcgagcg5360.3877.31Shimazoeeurofin
Z028TGAAAACTCATTACCTAAATTTGTTTATGTTCGGTAGCCCCTCGTTTAAACTGGATGGCGGCGTT6541.5471.34Shimazoeeurofin
Z029GTATCCTTTTCAAGTACTTCCACCACCACCACCATCACta404565.52Shimazoeeurofin
Z030taGTGATGGTGGTGGTGGTGGAAGTACTTGAAAAGGATAC404565.52Shimazoeeurofin
Z031caacctcaatggagtgatgcaacc245058.35Shimazoeeurofin
Z032AACAGGTGGATCCCACATTGtc225056.65Shimazoeeurofin
Z033CCTTTGCTCTGACCGATCCATA225056.03Shimazoeeurofin
Z034ACCCAGCACCATCAGAATTTAGCG245059.41Shimazoe eurofin
Z035CGGTGATTATCTAATCGAGGAAGAGG2646.1556.42Shimazoeeurofin
Z036GCTCATCAGTTCAATGGGAATCTTG254456.33Shimazoeeurofin
Z037CAGCTTTGCTGCTATGTATGGTG2347.8356.35Shimazoeeurofin
Z038GGAACCGCAAAACCAGACTAC2152.3855.81Shimazoeeurofin
Z039tttgtttgtttatgtgtgtttattcgaaac3026.6754.96Shimazoe eurofin
Z040AACAGGTGGATCCCACATTGtcatgtaatt304060.27Shimazoeeurofin
Z041gtttcgaataaacacacataaacaaacaaa3026.6754.96Shimazoeeurofin
Z042aattacatgaCAATGTGGGATCCACCTGTT304060.27Shimazoeeurofin
Z043GAAATTGCACTACCACCGGCGGCAAAATAT3046.6763.65Shimazoeeurofin
Z044 cctatgaactgatggttggtg 21 47.62 52.62 Shimazoe eurofin
Z045 ATACTGATGCTTCTGTAGAGGGTGA 25 44.00 56.62 Shimazoe eurofin
Z046 GATAGCTTGGAGTTCATCGCGAGT 24 50.00 58.77 Shimazoe eurofin
Z047 ggaagaggaggaagtgacatcgg 23 56.52 58.33 Shimazoe eurofin
Z048 ggcCAAACATCCTCACCCTCTAC 23 56.52 58.81 Shimazoe eurofin
Z049 ggacagttccatcgaggatc 20 55.00 53.93 Shimazoe eurofin
Z050 agcttcttttcttctgcgcc 20 50.00 55.58 Shimazoe eurofin
Z051 caggacgatggagtccaatg 20 55.00 54.50 Shimazoe eurofin
Z052 atatcggcaccttcgacctg 20 55.00 56.03 Shimazoe eurofin
Z053 GATGGACATAGGATCCTTAG 20 45.00 48.06 Shimazoe eurofin
Z054 GCCACATTGTCTTTTGTTGC 20 45.00 53.22 Shimazoe eurofin
Z055 CAGGACAGCGAGGCCGACTT 20 65.00 61.01 Shimazoe eurofin
Z056 GCAGTTGCCACTGGTTCCGA 20 60.00 59.69 Shimazoe eurofin
Z057 cgtatcgttgctcaaatgaa 20 40.00 51.00 Shimazoe eurofin
Z058 tcaccacctgttccggtggt 20 60.00 59.82 Shimazoe eurofin
Z059 GTAGAGTACAAGACGGCACT 20 50.00 53.16 Shimazoe eurofin
Z060 CAGCGTCCTACGATAATACG 20 50.00 52.27 Shimazoe eurofin
Z061 CCGGTTCTTCGTCCACTAAA 20 50.00 54.01 Shimazoe eurofin
Z062 CTATTTCCAGTGGAGAAACG 20 45.00 50.25 Shimazoe eurofin
Z063 ACAACAACCAGTTCGGAATC 20 45.00 52.73 Shimazoe eurofin
Z064 TAGATCCCACCACCAACACGGCC 23 60.87 62.47 Shimazoe eurofin
Z065 AGGCTATTACTGGTTATGGTCTTGG 25 44.00 55.94 Shimazoe eurofin
Z066 agttgataatttcatcttaatagagc 26 26.92 49.61 Shimazoe eurofin
Z067 acttcctcctcttccatc 18 50.00 49.02 Shimazoe eurofin
Z068 GGATGATTTGCGTTATTGTTCTTAT 25 32.00 52.46 Shimazoe eurofin
Z069 TTTCTCAAGCATGGGAAACAAGAGA 25 40.00 56.71 Shimazoe eurofin
Z070 GCCATGGAAGACCCACCAAGA 21 57.14 58.53 Shimazoe eurofin
Z071 ACGGCCAAAGAGAGCCATGG 20 60.00 59.19 Shimazoe eurofin
Z072 GTCTTGGTGGGTCTTCCATG 20 55.00 54.58 Shimazoe eurofin
Z073 GCGTTTCGAGGTCTTTGTAGCT 22 50.00 57.40 Shimazoe eurofin
Z074 TCTACAGAAGCATCAGTATC 20 40.00 48.01 Shimazoe eurofin
Z075 tcaatgtggttggttcagtgac 22 45.45 55.22 Shimazoe eurofin
Z076 agatccatcccttctcgatgtc 22 50.00 55.22 Shimazoe eurofin
Z077 GATGAAGTCGCTCTAGGTTGGG 22 54.55 56.66 Shimazoe eurofin
Z078 ACACACGAATTCGCGGCCGCTTCTAGATGGAGCAAGGTAGTAATGTGAATCACCTG 56 50.00 72.76 Nambu eurofin
Z079 CCCCACCTGCAGCGGCCGCTACTAGTATTATTATGTTTTTTCCGGTGGTAAATCTCCCTG 60 50.00 72.70 Nambu eurofin
Z080 CACACAGAATTCGCGGCCGCTTCTAGATGAATGAAGAGGGCTTTTTTTTCAGTGCCAGAGGCCACCGTCCT 71 52.11 76.39 Nambu eurofin
Z081 AAAATCACTGCAGCGGCCGCTACTAGTATTATTAGTCCAGAGGACGGTGGCCTCTGGCACTG 62 53.23 74.83 Nambu eurofin
Z082 GAAAAAGAATTCGCGGCCGCTTCTAGATGCAAACATCCTCACCCTCTACAGAAGC 55 49.09 71.86 Nambu eurofin
Z083 AGGGGGCTGCAGCGGCCGCTACTAGTATTATTACTTTGTACTAGGTGATGATGCAACAC 59 49.15 72.50 Nambu eurofin
Z084 GAACTCCAAGCTATCTGATC 20 45.00 49.40 Nambu eurofin
Z085 TGCCGACAGGAGAACagttgataatttcatcttaatagagc 41 39.02 63.55 Nambu eurofin
Z086 GTTCTCCTGTCGGCAggtttagcctcgactacttt 35 51.43 66.58 Shimazoe eurofin
Z087 TGCTGAAGTTGAATTggaaaattgggccaaagtag 35 40.00 63.03 Shimazoe eurofin
Z088 AATTCAACTTCAGCAtcttccaccgatgtcacttc 35 42.86 63.62 Shimazoe eurofin
Z089 GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGGGAACGAATGAAGAGGGCTTTTTTTTCAGTG 70 45.71 72.07 Shimazoe eurofin
Z090 TTCTGTAGAGGGTGAGGATGTTTGTTAGGCTAGCTTACTCGCGATGA 47 46.81 69.00 Shimazoe eurofin
Z091 GGATCCTAATACGACTCACTATAGGGAACAGCCACCATGGGCACAGAGCAAGGTAGTAATG 61 49.18 71.68 Shimazoe eurofin
Z092 CTGAAAAAGTACTTCTAGGCTCCCTTAGTGATGATGGTGATGGTGTCTGGA 51 45.10 68.47 Shimazoe eurofin
Z093 ccGGCTAGCTTACTCGCGATGAA 23 56.52 60.84 Shimazoe eurofin
Z094 ATGCATGAATTCGCGGCCGCTTCTAGAGACACGCTTTTTCAGTTCGAGTT 50 48.00 72.04 Nambu eurofin
Z095 TCCCCCCTGCAGCGGCCGCTACTAGTAGGCCGCAAATTA 39 61.54 74.13 Nambu eurofin
Z096 ATGGCTATCTGGGAGCAACT 20 50.00 54.79 Tong eurofin
Z097 ATCTACTGTCTGCAGAGTCAG 21 47.62 52.87 Tong eurofin
Z098 TACGTAGGATCCCTGCAGTC 20 55.00 54.71 Tong eurofin
Z099 GACTAGCAGGAGGTGAAGAT 20 50.00 52.43 Tong eurofin
Z100 TGAAAGCGAAGACGAGATGG 20 50.00 54.49 Tong eurofin
Z101 AGAGCTTTAACAGCAGTAGG 20 45.00 51.22 Tong eurofin
VR attaccgcctttgagtgagc 20 50.00 54.70 Tong eurofin
VF2 tgccacctgacgtctaagaa 20 50.00 55.05 Tong eurofin
Y001 ctaacggtacttctactgaag 21 42.86 49.31 Tong eurofin



3)Materials
3-1 Kit
NameSupplier
FastGene™Plasmid Mini KitNIPPON Genetics Co., Ltd
FastGene™Gel/PCR Extraction Kit NIPPON Genetics Co.,Ltd
3-2 Restriction Enzyme
NameSupplier
EcoRITaKaRa, Promega
PstITaKaRa, Promega
SpeITaKaRa, Promega
3-3 Polymerase
NameSupplier
Q5 High-Fidelity 2x master mixNew England Biolabs Japan Inc.
3-4 DNA ligase
NameSupplier
T4 DNA ligaseTaKaRa
3-5 Marker
NameSupplier
1kb DNA LadderNIPPON Genetics Co., Ltd
3-6 Organism
NameSupplier
E.coli DH5α GenotypeTaKaRa
3-7 Antibiotics
NameSupplier
ChloramphenicolWako
AmpicillinWako
3-8 Equipment
Name Supplier
BioPhotometereppendorf
LABO SHAKERBIO CRAFT
CO2 INCUBATORSANYO
Pipette ControllerBiohit Midi Plus
MiniCentrifuge Model GMC-060LMS CO.,LTD.
HIGH-SPEED REFRIGERATED CENTRIFUGETOMY
HIGH-PRESSURE STEAM STERILIZERTOMY
VOTEX-GENE2Scientific Industryes
Scanning Electron Miniscope TM1000sHITACHI
Fluorescence Microscope BX61N-34-FL-1-DOLYMPUS
SCIECE IMAGING SYSTEM LAS-3000Fuji film
3-9 Backbone
NameSupplier
pRS421National BioResource Project
http://yeast.nig.ac.jp/yeast/
pRS423 National BioResource Project
pRS424National BioResource Project
pRS425 National BioResource Project
pRS426National BioResource Project
pGK421National BioResource Project
pRS316 National BioResource Project
3-10 Buffer
NameSupplier
Sodium CarbonateWako
Sodium BicarbonateWako
Methanol(99.5%)Wako
Sodium ChlorideWako
SDSnacalai tesque
Trisaminomethanenacalai tesque
Potassium dihydrogenphosphsteSIGAMA-ALDRICH
Potassium ChlorideWako
GlycineWako
Agar, PowderWako
Agarose XPWako
10% Hydrochloric AcidWako
3-11 SDS-PAGE&Westernblotting
NameSupplier
REAL GEL PLATE (10%)BIO CRAFT
BE-210(SDS-PAGE)BIO CRAFT
BE-330BIO CRAFT
Amersham ECL Anti-Mouse IgGGE healthcare
Amersham ECL Anti-rabbit IgGGE healthcare
Amersham ECL Prime Blocking ReagentGE healthcare
Amersham ECL Prime WB Detection ReagentGE healthcare



4)Methods
4-1 Miniprep

Minipreps were performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

4-2 Gel Extraction and PCR purification

Gel Extraction was performed using FastGene™Gel/PCR Extraction Kit according to the manufacturer's protocols.

4-3 Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Q5 High-Fidelity 2x master mix according to the respective manufacturer's protocols.

4-4 Ligation

Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's.

4-5 Transformation

1.Thaw competent cells on ice
2.Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes
3.Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
4.Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37℃
5.Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37℃

4-6 PCR

PCR was performed using Q5 High-Fidelity 2x master mix according to the manufacturer's protocols.

4-7 Sequencing

We outsourced the sequencing to Macrogen

http://www.macrogen-japan.co.jp/cap_seq_0203.php

4-8 Western blotting

1.Apply sample to SDS-PAGE minigel (BIOCRAFT 15%).
2.Soak the gel in transfer buffer.
3.Soak PVDF membrane in 100% methanol for 30 sec.
4.Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min.
5.Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode.
6.Transfer the proteins from the gel to the membrane with 100 mA for 1 h.
7.Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h.
8.Wash for 5 min 3 times with TBST.
9.Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h.
10.Wash for 5 min 3 times with TBST.
11.Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h.
12.Wash for 5 min 3 times with TBST.
13.Drain excess wash buffer from the washed membrane and place on flat surface, protein side up.
14.Add detection reagent onto the membrane, covering all of the membrane.
15.Incubate for 5 minutes at room temperature.
16.Drain off excess detection reagent by dabbing with Kimwipe.
17.Place the sample in the CCD camera compartment and record the images.

4-9 Overnight-Culture

1.Prepare LB media by resolving 5g Bacto Tryptone, 1.25g Bacto-yeast extract and 1.25g NaCl in 500ml of milliQ and autoclave it.
2.Transfer 7ml of LB into a culture tube and add the respective antibiotic stock solutions.
3.Inoculate the culture by picking a single colony with a pipet tip and tipping it into the medium.
4.Shake the culture at 37℃ at 180 rpm for 14-16h.
5.Isolate plasmids or use the culture for further experiments.

Concentrations of antibiotics:
AntibioticsStock concentrationFinal concentration
Ampicillin100mg/ml100μg/ml
Chloramphenicol10mg/l30μg/ml

4-10 Plates

1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
2. Transfer 1. to the flask.
3. Place the following in the flask of 2.
・Bacto Tryptone 5 g
・Bacto yeast extract 2.5 g
・NaCl 2.5g
・Agar 7.5 g
4. After mixing thoroughly, autoclave it after covering with aluminum foil.
5. Add the respective antibiotics.
6. Cool until it gets moderately warm to the touch.
7. Put in a container and fix the lid with the tape after the medium solidifies.

Concentrations of antibiotics:
AntibioticsStock concentrationFinal concentration
Ampicillin100mg/ml100μg/ml
Chloramphenicol10mg/ml30μg/ml