|
|
Line 18: |
Line 18: |
| <tr> | | <tr> |
| <td> | | <td> |
- | <div id="sticky_cal_wrap"> | + | <!-- leave blank --> |
- | <div id="cal">
| + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"><a href="https://2014.igem.org/Team:Duke/Notebook#apr"> April 2014 </a></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7"><i> Month 1 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>sun</td>
| + | |
- | <td>mon</td>
| + | |
- | <td>tue</td>
| + | |
- | <td>wed</td>
| + | |
- | <td>thu</td>
| + | |
- | <td>fri</td>
| + | |
- | <td>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td>1</td>
| + | |
- | <td>2</td>
| + | |
- | <td>3</td>
| + | |
- | <td>4</td>
| + | |
- | <td>5</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>6</td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr7">7</a></td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr8">8</a></td>
| + | |
- | <td>9</td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr10">10</a></td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr11">11</a></td>
| + | |
- | <td>12</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr13">13</a></td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr14">14</a></td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr15">15</a></td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#apr16">16</a></td>
| + | |
- | <td>17</td>
| + | |
- | <td>18</td>
| + | |
- | <td>19</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>20</td>
| + | |
- | <td>21</td>
| + | |
- | <td>22</td>
| + | |
- | <td>23</td>
| + | |
- | <td>24</td>
| + | |
- | <td>25</td>
| + | |
- | <td>26</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>27</td>
| + | |
- | <td>28</td>
| + | |
- | <td>29</td>
| + | |
- | <td>30</td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> May 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 2 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>sun</td>
| + | |
- | <td>mon</td>
| + | |
- | <td>tue</td>
| + | |
- | <td>wed</td>
| + | |
- | <td>thu</td>
| + | |
- | <td>fri</td>
| + | |
- | <td>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td>1</td>
| + | |
- | <td>2</td>
| + | |
- | <td>3</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>4</td>
| + | |
- | <td>5</td>
| + | |
- | <td>6</td>
| + | |
- | <td>7</td>
| + | |
- | <td>8</td>
| + | |
- | <td>9</td>
| + | |
- | <td>10</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>11</td>
| + | |
- | <td>12</td>
| + | |
- | <td>13</td>
| + | |
- | <td>14</td>
| + | |
- | <td>15</td>
| + | |
- | <td>16</td>
| + | |
- | <td>17</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>18</td>
| + | |
- | <td>19</td>
| + | |
- | <td>20</td>
| + | |
- | <td>21</td>
| + | |
- | <td>22</td>
| + | |
- | <td>23</td>
| + | |
- | <td>24</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td>25</td>
| + | |
- | <td>26</td>
| + | |
- | <td>27</td>
| + | |
- | <td>28</td>
| + | |
- | <td><a href="https://2014.igem.org/Team:Duke/Notebook#may29">29</a></td>
| + | |
- | <td>30</td>
| + | |
- | <td>31</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | <td></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> June 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 3 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>sun</td>
| + | |
- | <td align=center>mon</td>
| + | |
- | <td align=center>tue</td>
| + | |
- | <td align=center>wed</td>
| + | |
- | <td align=center>thu</td>
| + | |
- | <td align=center>fri</td>
| + | |
- | <td align=center>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun1">1</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun2">2</a></td>
| + | |
- | <td align=center>3</td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun4">4</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun5">5</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun6">6</a></td>
| + | |
- | <td align=center>7</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>8</td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun9">9</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun10">10</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun11">11</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun12">12</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun13">13</a></td>
| + | |
- | <td align=center>14</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun15">15</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun16">16</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun17">17</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun18">18</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun19">19</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun20">20</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun21">21</a></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun22">22</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun23">23</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun24">24</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun25">25</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun26">26</a></td>
| + | |
- | <td align=center>27</td>
| + | |
- | <td align=center>28</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>29</td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jun30">30</a></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> July 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 4 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>sun</td>
| + | |
- | <td align=center>mon</td>
| + | |
- | <td align=center>tue</td>
| + | |
- | <td align=center>wed</td>
| + | |
- | <td align=center>thu</td>
| + | |
- | <td align=center>fri</td>
| + | |
- | <td align=center>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul1">1</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul2">2</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul3">3</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul4">4</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul5">5</a></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul6">6</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul7">7</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul8">8</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul9">9</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul10">10</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul11">11</a></td>
| + | |
- | <td align=center>12</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>13</td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul14">14</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul15">15</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul16">16</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul17">17</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul18">18</a></td>
| + | |
- | <td align=center>19</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul20">20</a></td>
| + | |
- | <td align=center><a href="https://2014.igem.org/Team:Duke/Notebook#jul21">21</a></td>
| + | |
- | <td align=center>22</td>
| + | |
- | <td align=center>23</td>
| + | |
- | <td align=center>24</td>
| + | |
- | <td align=center>25</td>
| + | |
- | <td align=center>26</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>27</td>
| + | |
- | <td align=center>28</td>
| + | |
- | <td align=center>29</td>
| + | |
- | <td align=center>30</td>
| + | |
- | <td align=center>31</td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> August 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 5 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>sun</td>
| + | |
- | <td align=center>mon</td>
| + | |
- | <td align=center>tue</td>
| + | |
- | <td align=center>wed</td>
| + | |
- | <td align=center>thu</td>
| + | |
- | <td align=center>fri</td>
| + | |
- | <td align=center>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center>1</td>
| + | |
- | <td align=center>2</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>3</td>
| + | |
- | <td align=center>4</td>
| + | |
- | <td align=center>5</td>
| + | |
- | <td align=center>6</td>
| + | |
- | <td align=center>7</td>
| + | |
- | <td align=center>8</td>
| + | |
- | <td align=center>9</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>10</td>
| + | |
- | <td align=center>11</td>
| + | |
- | <td align=center>12</td>
| + | |
- | <td align=center>13</td>
| + | |
- | <td align=center>14</td>
| + | |
- | <td align=center>15</td>
| + | |
- | <td align=center>16</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>17</td>
| + | |
- | <td align=center>18</td>
| + | |
- | <td align=center>19</td>
| + | |
- | <td align=center>20</td>
| + | |
- | <td align=center>21</td>
| + | |
- | <td align=center>22</td>
| + | |
- | <td align=center>23</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>24</td>
| + | |
- | <td align=center>25</td>
| + | |
- | <td align=center>26</td>
| + | |
- | <td align=center>27</td>
| + | |
- | <td align=center>28</td>
| + | |
- | <td align=center>29</td>
| + | |
- | <td align=center>30</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>31</td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> September 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 6 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>sun</td>
| + | |
- | <td align=center>mon</td>
| + | |
- | <td align=center>tue</td>
| + | |
- | <td align=center>wed</td>
| + | |
- | <td align=center>thu</td>
| + | |
- | <td align=center>fri</td>
| + | |
- | <td align=center>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center>1</td>
| + | |
- | <td align=center>2</td>
| + | |
- | <td align=center>3</td>
| + | |
- | <td align=center>4</td>
| + | |
- | <td align=center>5</td>
| + | |
- | <td align=center>6</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>7</td>
| + | |
- | <td align=center>8</td>
| + | |
- | <td align=center>9</td>
| + | |
- | <td align=center>10</td>
| + | |
- | <td align=center>11</td>
| + | |
- | <td align=center>12</td>
| + | |
- | <td align=center>13</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>14</td>
| + | |
- | <td align=center>15</td>
| + | |
- | <td align=center>16</td>
| + | |
- | <td align=center>17</td>
| + | |
- | <td align=center>18</td>
| + | |
- | <td align=center>19</td>
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- | <td align=center>20</td>
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- | </tr>
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- | <tr>
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- | <td align=center>21</td>
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- | <td align=center>22</td>
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- | <td align=center>23</td>
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- | <td align=center>24</td>
| + | |
- | <td align=center>25</td>
| + | |
- | <td align=center>26</td>
| + | |
- | <td align=center>27</td>
| + | |
- | </tr>
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- | <tr>
| + | |
- | <td align=center>28</td>
| + | |
- | <td align=center>29</td>
| + | |
- | <td align=center>30</td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
| + | |
- | </tr>
| + | |
- | </table>
| + | |
- | | + | |
- | <table>
| + | |
- | <tr>
| + | |
- | <td colspan="7"> October 2014 </td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td colspan="7" align=center><i> Month 7 of Project </i></td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>sun</td>
| + | |
- | <td align=center>mon</td>
| + | |
- | <td align=center>tue</td>
| + | |
- | <td align=center>wed</td>
| + | |
- | <td align=center>thu</td>
| + | |
- | <td align=center>fri</td>
| + | |
- | <td align=center>sat</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center></td>
| + | |
- | <td align=center>1</td>
| + | |
- | <td align=center>2</td>
| + | |
- | <td align=center>3</td>
| + | |
- | <td align=center>4</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>5</td>
| + | |
- | <td align=center>6</td>
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- | <td align=center>7</td>
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- | <td align=center>8</td>
| + | |
- | <td align=center>9</td>
| + | |
- | <td align=center>10</td>
| + | |
- | <td align=center>11</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>12</td>
| + | |
- | <td align=center>13</td>
| + | |
- | <td align=center>14</td>
| + | |
- | <td align=center>15</td>
| + | |
- | <td align=center>16</td>
| + | |
- | <td align=center>17</td>
| + | |
- | <td align=center>18</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>19</td>
| + | |
- | <td align=center>20</td>
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- | <td align=center>21</td>
| + | |
- | <td align=center>22</td>
| + | |
- | <td align=center>23</td>
| + | |
- | <td align=center>24</td>
| + | |
- | <td align=center>25</td>
| + | |
- | </tr>
| + | |
- | <tr>
| + | |
- | <td align=center>26</td>
| + | |
- | <td align=center>27</td>
| + | |
- | <td align=center>28</td>
| + | |
- | <td align=center>29</td>
| + | |
- | <td align=center>30</td>
| + | |
- | <td align=center>31</td>
| + | |
- | <td align=center></td>
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- | </tr>
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- | <tr>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | <td align=center></td>
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- | </tr>
| + | |
- | </table>
| + | |
- | </div>
| + | |
- | </div>
| + | |
| | | |
| </td> | | </td> |
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April Overview
Objectives
This is what we were trying to accomplish in April.
April 7
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw
PCR of Z4EV from pMN10
- new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM)
- 4 tubes with 0, 0.3, 0.6, 1 uL template
- iGEM 3-step protocol with 65C anneal, 20 sec extend
- Note: did not dilute template beforehand (do this in future)
Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)
- Expected band size 715 bp
- Control (no template) showed no band, lane 4 produced faint band
- Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)
Next steps:
- Run on gel to confirm (4/8)
- digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8)
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano
Agarose Gel with Z4EV PCR products
- PCR from 04/07/2014 of Z4EV from pMN10
- Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)
- Expected band size 715 bp
- Control (no template) showed no band, lane 4 produced faint band
- Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)
PCR cleanup of Z4EV PCR products
- PCR from 04/07/2014 of Z4EV from pMN10
- Used only lanes 2 and 3 (successful from gel)
- Concentration 32.4 ng/uL in 50 uL
Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1
- Both with SpeI-HF and NcoI-HF in Cutsmart
- PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction
- dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction
PCR cleanup of Z4EV PCR digest product
- 04/08 digest of 04/07 PCR
- Results: Final concentration negligible, no DNA
Next steps:
- Z4EV PCR again from pMN10
- Digest of PCR in SpeI/NcoI
- Gel extraction of digested TDH3-dCas9-MxiI
April 10
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano
PCR of Z4EV from pMN10
- Repeat of PCR from 4/7/14
- Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p
- Diluted pMN10 template to 1 ng/uL before use
- 8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction
- iGEM 3-step protocol with 65C anneal, 20 sec extend
- Results (see 4/11): expected bands present, but concentrations too low
Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest
- Digest performed 4/8 with SpeI/NcoI
- Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)
- Obtained consistent band sizes, used 400 mg gel in extraction <
- Gel picture:
- Results: obtained 20 uL product at 55 ng/uL (froze for later use)
Next steps:
- Run gel of PCR
- Clean up PCR
April 11
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw
Agarose gel to analyze PCR of Z4EV from pMN10
- PCR from 4/10
- Expected band size: 715 bp
- Showed band at expected size in all seven non-control lanes
- Intensities appear low, increase in higher lanes (higher template conc.)
- Gel picture:
PCR cleanup of Z4EV from 4/10 PCR
- final concentration from 7 tubes: 25.8 ng/uL in 30 uL
- Need to improve
Next Steps:
- Redo PCR of Z4EV. Things to try:
- Use previous cleanup as a template
- use 1 uL enzyme instead of 0.5
- slightly longer extension time
- higher template concentration
- PCR cleanup: Things to try:
- Run PB buffer flow-through back through filter during step 7
- let PE buffer sit in column (covered) while ethanol evaporates
- let elution buffer sit on DNA for 2-3 minutes before spin
April 13 - Unfinished
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Garima Tomar
PCR of Z4EV from pMN10
- Third attempt (previously 4/7, 4/10)
- oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p
- 8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction
- Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction
- iGEM 3-step protocol with 65C anneal, 1 min extension
- Changes from previous: more template, more polymerase, longer extension
Results (Date):
Next steps:
- Run on gel
- Clean up PCR with new notes (see 4/11), see if concentration is better
April 14
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano
Agarose gel of Z4EV PCR
- Z4EV from pMN10, 4/13 PCR
- 2-log ladder and 8 identical reaction tubes
- Expected band size ~715 bp
- All lanes showed band 700-800 bp
- band intensity much greater than previous attempts
- Unable to take normal picture: used dark room and iPhone camera to photograph
PCR cleanup of Z4EV PCR from 4/13
- Qiagen miniprep steps 7-10
- 8 tubes into 1 final product
- Modifications to protocol:
- ran PB buffer through twice
- let column sit, covered, for 10-15 min after PE buffer wash/discard step
- let water for elution sit on column for 3 min before spin
- Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use)
Next Steps:
- Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI
- ligation of Z4EV with dCas9 backbone (obtained 4/10)
- transformation
April 15
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano
Restriction Digest of Z4EV-PCR with SpeI/NcoI
- PCR from 4/13, cleaned 4/14
- Cut with SpeI-HF and NcoI-HF in Cutsmart buffer
- 30 uL PCR product (407.5 ng/uL) in 50 uL reaction
- Incubated at 37C for 4.5 hrs
PCR Cleanup of Restriction Digest of Z4EV
- Qiagen kit with modified methods described on 4/14
- Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop)
Ligation of Z4EV PCR and dCas9-MxiI backbone
- Z4EV from pMN10, digested with NcoI and SpeI (4/15)
- dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10
- 3:1 molar ratio backbone:insert
- 20 uL total reaction - 15.3 uL backbone (55 ng/uL), 0.7 uL insert (248.1 ng/uL)
- Left in cold room overnight in 16C heat block
Next steps:
- Transform ligated plasmid into E. coli, clone, then miniprep.
April 16
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Charlie Cooper
Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli
- Ligation left overnight for 22 hrs
- Heat shock protocol with chemically competent E. coli
- 1 sample plus 1 no-insert control
- grown in SOC medium, plated on LB+Amp plates
- Results (Charlie Cooper) (4/17): No colonies on either plate
- Possibly used ineffective cells
Next steps:
- Digest and gel extraction of backbone
- Ligation and Transformation
May 29
Objective: Prepare Chemically competent E. Coli
Matthew Faw
Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli
- Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left
Next Steps:
June 1
Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw
Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA
- Followed Charlie’s Cloning Protocols
- Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
- Plated the transformed DNA and put in incubator at 37C
- Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw
Next Steps:
- Look at plates, compare cultures
June 2
Objective: Redo yesterday’s transformation
Matthew Faw
Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.
- Followed Charlie’s Cloning Protocols
- Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
- Plated the transformed DNA on 6 separate plates, put in 37C overnight
- Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol
Next Steps:
- Look at plates, compare cultures, etc.
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