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| | color:#000000; | | color:#000000; |
| | border-collapse:collapse; | | border-collapse:collapse; |
| − | border-left:1px solid #000000; | + | border: solid 3px #25b6ca; |
| − | border-right:1px solid #000000;
| + | |
| − | border-top:1px solid #000000;
| + | |
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| | | | |
| | th{ | | th{ |
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| − | border-bottom:1px solid #ffffff;
| + | |
| | background-color:#25b6ca; | | background-color:#25b6ca; |
| | + | height:10px; |
| | color:#ffffff; | | color:#ffffff; |
| | } | | } |
| | | | |
| | td{ | | td{ |
| − | border-right:1px solid #000000; | + | border-right:3px solid #000000; |
| − | border-bottom:1px solid #000000; | + | border-bottom:3px solid #000000; |
| | + | border: solid 3px #25b6ca; |
| | + | background-color:#ffffff; |
| | word-wrap:break-word; | | word-wrap:break-word; |
| | + | color:#757575; |
| | } | | } |
| | | | |
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| | <div id="wrapper"><br><br><br> | | <div id="wrapper"><br><br><br> |
| | <h1><font face="Segoe UI">Basic Parts</font></h1> | | <h1><font face="Segoe UI">Basic Parts</font></h1> |
| − | <ul class="material">
| |
| − | <li><a href="#Parts"><font face="Segoe UI">1) Parts</font></a></li>
| |
| − | <li><a href="#Primer"><font face="Segoe UI">2) Primer list</font></a></li>
| |
| − | <li><a href="#Materials"><font face="Segoe UI">3) Materials</font></a></li>
| |
| − | <ul>
| |
| − | <li><a href="#Kit"><font face="Segoe UI">3-1 Kit</font></a></li>
| |
| − | <li><a href="#Restriction Enzyme"><font face="Segoe UI">3-2 Restriction Enzyme</font></a></li>
| |
| − | <li><a href="#Polymerase"><font face="Segoe UI">3-3 Polymerase</font></a></li>
| |
| − | <li><a href="#DNA ligase"><font face="Segoe UI">3-4 DNA ligase</font></a></li>
| |
| − | <li><a href="#Marker"><font face="Segoe UI">3-5 Marker</font></a></li>
| |
| − | <li><a href="#Organism"><font face="Segoe UI">3-6 Organism</font></a></li>
| |
| − | <li><a href="#Antibiotics"><font face="Segoe UI">3-7 Antibiotics</font></a></li>
| |
| − | <li><a href="#Equipment"><font face="Segoe UI">3-8 Equipment</font></a></li>
| |
| − | <li><a href="#Backbone"><font face="Segoe UI">3-9 Backbone</font></a></li>
| |
| − | <li><a href="#Buffer"><font face="Segoe UI">3-10 Buffer</font></a></li>
| |
| − | <li><a href="#SDS-PAGE&Westernblotting"><font face="Segoe UI">3-11 SDS-PAGE&Westernblotting</font></a></li>
| |
| − | <!--------------------------------------------------------- BLOCK Contents-2 Materials END----------------------------------------------------------->
| |
| − | </ul>
| |
| − | <li><a href="#methods"><font face="Segoe UI">4) Methods</font></a></li>
| |
| − | <ul>
| |
| | | | |
| − | <!--------------------------------------------------------- BLOCK Contents-3 Methods PY------------------------------------------------------------>
| + | <!-- Table Generated by TableMaker.py Author: OHAD --> |
| − | <li><a href="#Miniprep"><font face="Segoe UI">4-1 Miniprep</font></a></li>
| + | <h6 id = "Buffer"><font face="Segoe UI">Basic Parts</font></h6> |
| − | <li><a href="#Gel Extraction and PCR purification"><font face="Segoe UI">4-2 Gel Extraction and PCR purification</font></a></li>
| + | <table> |
| − | <li><a href="#Restriction Enzyme Digestion"><font face="Segoe UI">4-3 Restriction Enzyme Digestion</font></a></li>
| + | <tr><th>Number |
| − | <li><a href="#Ligation"><font face="Segoe UI">4-4 Ligation</font></a></li>
| + | </th><th>Parts |
| − | <li><a href="#Transformation"><font face="Segoe UI">4-5 Transformation</font></a></li>
| + | </th><th>Designer |
| − | <li><a href="#PCR"><font face="Segoe UI">4-6 PCR</font></a></li>
| + | </th><th>Links |
| − | <li><a href="#Sequencing"><font face="Segoe UI">4-7 Sequencing</font></a></li>
| + | </th><th>用意すべき実験データ |
| − | <li><a href="#Western blotting"><font face="Segoe UI">4-8 Western blotting</font></a></li>
| + | </th><th>提出 |
| − | <li><a href="#Overnight-Culture"><font face="Segoe UI">4-9 Overnight-Culture</font></a></li>
| + | </th><th>ページ編集 |
| − | <li><a href="#Plates"><font face="Segoe UI">4-10 Plates</font></a></li>
| + | </th><th>備考 |
| − | <!--------------------------------------------------------- BLOCK Contents-3 Methods END------------------------------------------------------------>
| + | </th></tr> |
| − | </ul>
| + | |
| − | </ul>
| + | |
| − | <!-- ###################################################### BLOCK Contents END #################################################-->
| + | |
| − | | + | |
| − | <br><br><br>
| + | |
| − | | + | |
| − | | + | |
| − | <!-----------------------------------------------------BLOCK Parts-------------------------------------------------------------->
| + | |
| − | <h5 id="Parts"><font face="Segoe UI">1) Parts</font></h5>
| + | |
| − | <h6><a href="https://2018.igem.org/Team:Kyoto/Basic_Part" target="brank"><font face="Segoe UI">Basic Parts</font></a></h6>
| + | |
| − | <h6><a href="https://2018.igem.org/Team:Kyoto/Composite_Parts" target="brank"><font face="Segoe UI">Composite Parts</font></a></h6><br><br><br>
| + | |
| − | | + | |
| − | <!-----------------------------------------------------BLOCK Parts END---------------------------------------------------------->
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <!------------------------------------------------------BLOCK primer PY----------------------------------------------------------><!-- Table Generated by TableMaker.py Author: OHAD -->
| + | |
| − | <h5 id = "Primer"><font face="Segoe UI">2) Primer List</font></h5> | + | |
| − | <div class="example">
| + | |
| − | <table width="20px"> | + | |
| − | <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 | + | <tr><td> |
| − | </td><td>cctatgaactgatggttggtg | + | </td><td>BBa_K2665011 |
| − | </td><td>21 | + | </td><td>TDH3-Mangrin-6xHis-CYC |
| − | </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>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665011 |
| − | </td></tr><tr><td>Z097 | + | </td><td>スポットアッセイとボイル法 |
| − | </td><td>ATCTACTGTCTGCAGAGTCAG | + | </td><td>する、１A |
| − | </td><td>21 | + | </td><td>あとは実験データのせるだけ |
| − | </td><td>47.62 | + | </td><td>なし |
| − | </td><td>52.87 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665012 |
| | + | </td><td>TDH3-ZrGPD1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin</td></tr><tr><td>Z098 | + | </td><td>http://parts.igem.org/Part:BBa_K2665012 |
| − | </td><td>TACGTAGGATCCCTGCAGTC | + | </td><td>〃 |
| − | </td><td>20 | + | </td><td>する１B |
| − | </td><td>55.00 | + | </td><td>〃 |
| − | </td><td>54.71 | + | </td><td>なし |
| | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665013 |
| | + | </td><td>TDH3-AtNHXS1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665013 |
| − | </td></tr><tr><td>Z099 | + | </td><td>〃 |
| − | </td><td>GACTAGCAGGAGGTGAAGAT | + | </td><td>する１C |
| − | </td><td>20 | + | </td><td>〃 |
| − | </td><td>50.00 | + | </td><td>なし |
| − | </td><td>52.43 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665014 |
| | + | </td><td>TDH3-SseNHX1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665014 |
| − | </td></tr><tr><td>Z100 | + | </td><td>〃 |
| − | </td><td>TGAAAGCGAAGACGAGATGG | + | </td><td>する１E |
| − | </td><td>20 | + | </td><td>未完 |
| − | </td><td>50.00 | + | </td><td>codon optimized |
| − | </td><td>54.49 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665015 |
| | + | </td><td>TDH3-AVP1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665015 |
| − | </td></tr><tr><td>Z101 | + | </td><td>〃 |
| − | </td><td>AGAGCTTTAACAGCAGTAGG | + | </td><td>する１D |
| − | </td><td>20 | + | </td><td>未完 |
| − | </td><td>45.00 | + | </td><td>終止コドン消してないことを書く |
| − | </td><td>51.22 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665016 |
| | + | </td><td>TDH3-AtHKT1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665016 |
| − | </td></tr><tr><td>VR | + | </td><td>〃 |
| − | </td><td>attaccgcctttgagtgagc | + | </td><td>する１F |
| − | </td><td>20 | + | </td><td>未完 |
| − | </td><td>50.00 | + | </td><td>なし |
| − | </td><td>54.70 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665017 |
| | + | </td><td>TDH3-ZrFPS1-6xHis-CYC |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665017 |
| − | </td></tr><tr><td>VF2 | + | </td><td>〃 |
| − | </td><td>tgccacctgacgtctaagaa | + | </td><td>しない |
| − | </td><td>20 | + | </td><td>未完 |
| − | </td><td>50.00 | + | </td><td>なし |
| − | </td><td>55.05 | + | </td></tr><tr><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td><td> |
| | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665018 |
| | + | </td><td>Fgβ |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>http://parts.igem.org/Part:BBa_K2665018 |
| − | </td></tr><tr><td>Y001 | + | </td><td>蛍光顕微鏡 |
| − | </td><td>ctaacggtacttctactgaag | + | </td><td>する2A |
| − | </td><td>21 | + | </td><td>未完 |
| − | </td><td>42.86 | + | </td><td>なし |
| − | </td><td>49.31 | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665019 |
| | + | </td><td>SdrG |
| | </td><td>Tong | | </td><td>Tong |
| − | </td><td>eurofin | + | </td><td>〃 |
| | + | </td><td>する2B |
| | + | </td><td>未完 |
| | + | </td><td>なし |
| | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665022 |
| | + | </td><td>Stalk |
| | + | </td><td>Tong |
| | + | </td><td> |
| | + | </td><td>〃 |
| | + | </td><td>しない |
| | + | </td><td>未完 |
| | + | </td><td>なし |
| | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665023 |
| | + | </td><td>ybbr-tag |
| | + | </td><td>Tong |
| | + | </td><td> |
| | + | </td><td>K2665018と2665019を参照しろと書く |
| | + | </td><td>しない |
| | + | </td><td>未完 |
| | + | </td><td>なし |
| | + | </td></tr><tr><td> |
| | + | </td><td>BBa_K2665024 |
| | + | </td><td>ddFLN4 |
| | + | </td><td>Tong |
| | + | </td><td> |
| | + | </td><td>K2665018と2665020を参照しろと書く |
| | + | </td><td>しない |
| | + | </td><td>未完 |
| | + | </td><td>なし |
| | </td></tr> | | </td></tr> |
| | | | |
| Line 597: |
Line 203: |
| | | | |
| | | | |
| − |
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| − | </table>
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| − | </div>
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| − | <!-- Table end --><br><br><br>
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| − |
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| − |
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| − | <!------------------------------------------------------BLOCK primer.py END---------------------------------------------------------->
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| − |
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| − |
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| − |
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| − | <h5 id="Materials"><font face="Segoe UI">3) Materials</font></h5>
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| − |
| |
| − | <!------------------------------------------------------BLOCK TableMaker PY------------------------------------------------------>
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Kit"><font face="Segoe UI">3-1 Kit</font></h6>
| |
| − | <table>
| |
| − | <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 -->
| |
| − | <h6 id = "Restriction Enzyme"><font face="Segoe UI">3-2 Restriction Enzyme</font></h6>
| |
| − | <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>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Polymerase"><font face="Segoe UI">3-3 Polymerase</font></h6>
| |
| − | <table>
| |
| − | <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 end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "DNA ligase"><font face="Segoe UI">3-4 DNA ligase</font></h6>
| |
| − | <table>
| |
| − | <tr><th>Name</th><th>Supplier</th></tr>
| |
| − | <tr><td>T4 DNA ligase</td><td>TaKaRa</td></tr>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Marker"><font face="Segoe UI">3-5 Marker</font></h6>
| |
| − | <table>
| |
| − | <tr><th>Name</th><th>Supplier</th></tr>
| |
| − | <tr><td>1kb DNA Ladder</td><td>NIPPON Genetics Co., Ltd</td></tr>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Organism"><font face="Segoe UI">3-6 Organism</font></h6>
| |
| − | <table>
| |
| − | <tr><th>Name</th><th>Supplier</th></tr>
| |
| − | <tr><td>E.coli DH5α Genotype</td><td>TaKaRa</td></tr>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Antibiotics"><font face="Segoe UI">3-7 Antibiotics</font></h6>
| |
| − | <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>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Equipment"><font face="Segoe UI">3-8 Equipment</font></h6>
| |
| − | <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>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − |
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Backbone"><font face="Segoe UI">3-9 Backbone</font></h6>
| |
| − | <table>
| |
| − | <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 end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "Buffer"><font face="Segoe UI">3-10 Buffer</font></h6>
| |
| − | <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>
| |
| − | </table>
| |
| − | <!-- Table end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − | <h6 id = "SDS-PAGE&Westernblotting"><font face="Segoe UI">3-11 SDS-PAGE&Westernblotting</font></h6>
| |
| − | <table>
| |
| − | <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 end -->
| |
| − | <!-- Table Generated by TableMaker.py Author: OHAD -->
| |
| − |
| |
| − | <table>
| |
| − | <tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr><tr></tr>
| |
| − | </table>
| |
| − | <!-- Table end --><br><br><br>
| |
| − |
| |
| − |
| |
| − | <!------------------------------------------------------BLOCK TableMaker.py END------------------------------------------------------>
| |
| − |
| |
| | | | |
| | <h5 id="methods"><font face="Segoe UI">4) Methods</font></h5> | | <h5 id="methods"><font face="Segoe UI">4) Methods</font></h5> |
| Line 756: |
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| | </p> | | </p> |
| | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ --> | | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ --> |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Gel Extraction and PCR purification"><font face="Segoe UI">4-2 Gel Extraction and PCR purification</font></h6>
| |
| − | <p>
| |
| − | Gel Extraction was performed using FastGene™Gel/PCR Extraction Kit according to the manufacturer's protocols.
| |
| − | </p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Restriction Enzyme Digestion"><font face="Segoe UI">4-3 Restriction Enzyme Digestion</font></h6>
| |
| − | <p>
| |
| − | Restriction enzyme treatment was performed using Q5 High-Fidelity 2x master mix according to the respective manufacturer's protocols.
| |
| − | </p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Ligation"><font face="Segoe UI">4-4 Ligation</font></h6>
| |
| − | <p>
| |
| − | Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's.
| |
| − | </p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Transformation"><font face="Segoe UI">4-5 Transformation</font></h6>
| |
| − | <p>
| |
| − | 1.Thaw competent cells on ice<br>
| |
| − | 2.Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes</br>
| |
| − | 3.Keep them in heating block for 45 seconds, then cool on ice for 2 minutes<br>
| |
| − | 4.Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37℃<br>
| |
| − | 5.Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37℃
| |
| − | </p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "PCR"><font face="Segoe UI">4-6 PCR</font></h6>
| |
| − | <p>
| |
| − | PCR was performed using Q5 High-Fidelity 2x master mix according to the manufacturer's protocols.</p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Sequencing"><font face="Segoe UI">4-7 Sequencing</font></h6>
| |
| − | <p>
| |
| − | We outsourced the sequencing to Macrogen
| |
| − | </p>
| |
| − | <p>
| |
| − | http://www.macrogen-japan.co.jp/cap_seq_0203.php
| |
| − | </p>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − |
| |
| − |
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Western blotting"><font face="Segoe UI">4-8 Western blotting</font></h6>
| |
| − | <p>
| |
| − | 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>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Overnight-Culture"><font face="Segoe UI">4-9 Overnight-Culture</font> </h6>
| |
| − | <p>
| |
| − | 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>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
| |
| − |
| |
| − | <!-- ##############The below part of block is Generated by Methods.py Author: OHAD################# -->
| |
| − | <h6 id = "Plates"><font face="Segoe UI">4-10 Plates</font></h6>
| |
| − | <p>
| |
| − | 1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
| |
| − | <br>2. Transfer 1. to the flask.
| |
| − | <br>3. Place the following in the flask of 2.
| |
| − | <br> ・Bacto Tryptone 5 g
| |
| − | <br> ・Bacto yeast extract 2.5 g
| |
| − | <br> ・NaCl 2.5g
| |
| − | <br> ・Agar 7.5 g
| |
| − | <br>4. After mixing thoroughly, autoclave it after covering with aluminum foil.
| |
| − | <br>5. Add the respective antibiotics.
| |
| − | <br>6. Cool until it gets moderately warm to the touch.
| |
| − | <br>7. Put in a container and fix the lid with the tape after the medium solidifies.
| |
| − | </p>
| |
| − | <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/ml</td><td>30μg/ml</td></tr>
| |
| − | </table>
| |
| − | <!-- ~~~~~~~~~~~~~Part END~~~~~~~~~~~~~~ -->
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| | <!------------------------------------------------------BLOCK Methods.py END -----------------------------------------------------------> | | <!------------------------------------------------------BLOCK Methods.py END -----------------------------------------------------------> |
