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| | | |
| </section> | | </section> |
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| <header> | | <header> |
| <h2 class="inner-h">Notebook</h2> | | <h2 class="inner-h">Notebook</h2> |
− | <h3 class="inner-h">Notebook</h3>
| |
| | | |
− | <table> | + | <table style="width: auto;margin:auto"> |
− | <tbody valign="middle"> | + | <tbody> |
| <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> | | <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> |
| <td> </td> | | <td> </td> |
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| Got to Know RyR targeting antibody AR185. | | Got to Know RyR targeting antibody AR185. |
| </td> | | </td> |
− | <td rowspan="5";valign="middle"> | + | <td rowspan="5" valign="middle" text-align:"center";> |
| Cloning of AR185 began | | Cloning of AR185 began |
| <br> | | <br> |
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| </td> | | </td> |
| <td> | | <td> |
− | Collaborated with NUDT and (三军大队伍名字). | + | Collaborated with NUDT. |
| <br> | | <br> |
| Attended the 5th CCIC(Conference of China iGEMer Community). | | Attended the 5th CCIC(Conference of China iGEMer Community). |
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| </tr> | | </tr> |
| | | |
− | <tr> | + | <tr style="border-bottom: 1px #5d5d5d solid"> |
| <td>October 2018</td> | | <td>October 2018</td> |
| <td></td> | | <td></td> |
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| <section> | | <section> |
| <header> | | <header> |
− | <h3 class="inner-h">Protocol</h3> | + | <h2 class="inner-h">Protocol</h2> |
| </header> | | </header> |
| | | |
− | <h4 class="inner-h">Agarose Gel electrophoresis</h4> | + | <h3 class="inner-h">Agarose Gel electrophoresis</h3> |
| <p class="inner-text">1. Preparing the agarose gel</p> | | <p class="inner-text">1. Preparing the agarose gel</p> |
| <p class="inner-text"> | | <p class="inner-text"> |
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| </p> | | </p> |
| | | |
− | <h4 class="inner-h">PCR</h4> | + | <h3 class="inner-h">PCR</h3> |
| | | |
| <p class="inner-text"> | | <p class="inner-text"> |
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| <tbody valign="middle"> | | <tbody valign="middle"> |
| <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> | | <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> |
− | <td>Component</td> | + | <td width="300px">Component</td> |
− | <td>50 µL reaction</td> | + | <td width="300px">50 µL reaction</td> |
| </tr> | | </tr> |
| <tr> | | <tr> |
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| <td>About 200ng</td> | | <td>About 200ng</td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="border-bottom: 1px #5d5d5d solid"> |
| <td>ddH2O</td> | | <td>ddH2O</td> |
| <td>Up to 50</td> | | <td>Up to 50</td> |
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| <td></td> | | <td></td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="border-bottom: 1px #5d5d5d solid"> |
| <td>Hold</td> | | <td>Hold</td> |
| <td>4℃</td> | | <td>4℃</td> |
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| </table> | | </table> |
| | | |
− | <h4 class="inner-h">Preparation of LB Broth</h4> | + | <h3 class="inner-h">Preparation of LB Broth</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| 1. add 10g Trytone; | | 1. add 10g Trytone; |
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| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Transformation</h4> | + | <h3 class="inner-h">Transformation</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| 1. Put the tube of E. coli cells on ice until the last crystals disappear. And add 1-5 ul containing 1-100ng plasmid DNA to 50 ul of cells in a transformation tube on ice. | | 1. Put the tube of E. coli cells on ice until the last crystals disappear. And add 1-5 ul containing 1-100ng plasmid DNA to 50 ul of cells in a transformation tube on ice. |
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| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Gel extraction</h4> | + | <h3 class="inner-h">Gel extraction</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| Gel extraction was performed according to the Universal DNA purification Kit(Cat. DP214-03). | | Gel extraction was performed according to the Universal DNA purification Kit(Cat. DP214-03). |
| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Extraction and Purification of plasmid DNA</h4> | + | <h3 class="inner-h">Extraction and Purification of plasmid DNA</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| Plasmid extraction were carried out according to the GoldHi EndoFree Plasmid MAXI Kit. (Cat. CW2104M) for midiprep and TIANprep Mini Plasmid Kit.(Cat. DP103-03) for miniprep. | | Plasmid extraction were carried out according to the GoldHi EndoFree Plasmid MAXI Kit. (Cat. CW2104M) for midiprep and TIANprep Mini Plasmid Kit.(Cat. DP103-03) for miniprep. |
| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Site-Directed Mutagenesis</h4> | + | <h3 class="inner-h">Site-Directed Mutagenesis</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| We used forward and reverse primers which prime at the same site and contain a mismatch at the specific base in terms of the original structure. Each primer contains 25-30 bases. And this mismatch defines the new sequence of the same site. Like PCR, the designing mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation. And we do all the procedure according to the Fast Mutagenesis System Kit(Code#FM111-01). | | We used forward and reverse primers which prime at the same site and contain a mismatch at the specific base in terms of the original structure. Each primer contains 25-30 bases. And this mismatch defines the new sequence of the same site. Like PCR, the designing mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation. And we do all the procedure according to the Fast Mutagenesis System Kit(Code#FM111-01). |
| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Ligation</h4> | + | <h3 class="inner-h">Ligation</h3> |
| <table> | | <table> |
| <tbody valign="middle"> | | <tbody valign="middle"> |
| <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> | | <tr style="border-top: 1px #5d5d5d solid;border-bottom: 1px #5d5d5d solid"> |
− | <td>Component</td> | + | <td width="300px">Component</td> |
− | <td>Volume</td> | + | <td width="300px">Volume</td> |
| </tr> | | </tr> |
| <tr> | | <tr> |
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| <td>1 μL</td> | | <td>1 μL</td> |
| </tr> | | </tr> |
− | <tr> | + | <tr style="border-bottom: 1px #5d5d5d solid"> |
| <td>ddH2O</td> | | <td>ddH2O</td> |
| <td>Add to 20 μL</td> | | <td>Add to 20 μL</td> |
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| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Cell culture</h4> | + | <h3 class="inner-h">Cell culture</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| All of the procedure about cells must be carried out under sterile conditions. | | All of the procedure about cells must be carried out under sterile conditions. |
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| </p> | | </p> |
| | | |
− | <h4 class="inner-h">Establishing AAV-9</h4> | + | <h3 class="inner-h">Establishing AAV-9</h3> |
| <p class="inner-text"> | | <p class="inner-text"> |
| We used 293T cells to produce AAV-9. 293T cells express E1 gene products and they are commonly and extensively used for the production of E1-deleted AD viruses. All the procedure need to be done in biosafety cabinet. | | We used 293T cells to produce AAV-9. 293T cells express E1 gene products and they are commonly and extensively used for the production of E1-deleted AD viruses. All the procedure need to be done in biosafety cabinet. |
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| <h2>Wanna contact us?</h2> | | <h2>Wanna contact us?</h2> |
| </header> | | </header> |
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| <li> | | <li> |
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| <li> | | <li> |
| <h3>E-Mail</h3> | | <h3>E-Mail</h3> |
− | <p><a href="mailto:hus@smmu.edu.cn">hus@smmu.edu.cn</a></p> | + | <p><a href="mailto:igem-smmu@smmu.edu.cn">igem-smmu@smmu.edu.cn</a></p> |
| </li> | | </li> |
| <li> | | <li> |
| <h3>Phone</h3> | | <h3>Phone</h3> |
− | <p>+86 13701692043</p> | + | <p>+86 021-81870925</p> |
| </li> | | </li> |
| </ul> | | </ul> |
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| <div id="copyright"> | | <div id="copyright"> |
| <ul class="links"> | | <ul class="links"> |
− | <li>© Untitled. All rights reserved.</li> | + | <li>© 2018. SMMU. All rights reserved. Thanks to: <a href="http://html5up.net">HTML5 UP</a></li> |
− | <!-- <li>Design: <a href="http://html5up.net">HTML5 UP</a></li> -->
| + | |
| </ul> | | </ul> |
| </div> | | </div> |
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| }); | | }); |
| </script> | | </script> |
| + | |
| + | <script src="https://2018.igem.org/Template:SMMU-China/2019_JS_jquery_min?action=raw&ctype=text/javascript"></script> |
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| <p id="last_p_label"> | | <p id="last_p_label"> |
| </html> | | </html> |