Team:Duke/Notebook

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<p class="big"> Lab Notebook </p>
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Overview">Overview of Each Month </a></div>
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Months">Notebook Entries by Month</a></div>
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<div class="month"><a href="https://2014.igem.org/Team:Duke/Notebook/Protocols">Protocols</a></div>
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<td colspan="7"><a href="https://2014.igem.org/Team:Duke/Notebook#apr"> April 2014 </a></td>
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<td colspan="7"><i> Month 1 of Project </i></td>
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<td>sun</td>
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<td>1</td>
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<td>2</td>
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<td>3</td>
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<td>4</td>
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<td>5</td>
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<td>6</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr7">7</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr8">8</a></td>
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<td>9</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr10">10</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr11">11</a></td>
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<td>12</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr13">13</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr14">14</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr15">15</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#apr16">16</a></td>
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<td>17</td>
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<td>18</td>
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<td>19</td>
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<td>20</td>
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<td>21</td>
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<td>22</td>
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<td>23</td>
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<td>27</td>
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<td colspan="7"> May 2014 </td>
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<td colspan="7" align=center><i> Month 2 of Project </i></td>
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<td>sun</td>
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<td>mon</td>
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<td>1</td>
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<td>3</td>
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<td>4</td>
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<td>5</td>
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<td>6</td>
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<td>7</td>
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<td>8</td>
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<td>9</td>
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<td>10</td>
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<td>11</td>
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<td>12</td>
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<td>13</td>
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<td>14</td>
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<td>15</td>
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<td>16</td>
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<td>17</td>
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<td>18</td>
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<td>19</td>
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<td>20</td>
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<td>21</td>
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<td>22</td>
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<td>23</td>
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<td>24</td>
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<td>25</td>
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<td>26</td>
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<td>27</td>
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<td>28</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#may29">29</a></td>
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<td>30</td>
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<td>31</td>
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<td colspan="7"> June 2014 </td>
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<td colspan="7" align=center><i> Month 3 of Project </i></td>
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<td>sun</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun1">1</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun2">2</a></td>
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<td>3</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun4">4</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun5">5</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun6">6</a></td>
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<td>7</td>
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<td>8</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun9">9</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun10">10</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun11">11</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun12">12</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun13">13</a></td>
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<td>14</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun15">15</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun16">16</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun17">17</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun18">18</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun19">19</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun20">20</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun21">21</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun22">22</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun23">23</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun24">24</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun25">25</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun26">26</a></td>
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<td>27</td>
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<td>28</td>
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<td>29</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun30">30</a></td>
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<td colspan="7"> July 2014 </td>
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<td colspan="7" align=center><i> Month 4 of Project </i></td>
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<td>sun</td>
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<td>mon</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul1">1</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul2">2</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul3">3</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul4">4</a></td>
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<td ><a href="https://2014.igem.org/Team:Duke/Notebook#jul5">5</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul6">6</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul7">7</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul8">8</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul9">9</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul10">10</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul11">11</a></td>
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<td>12</td>
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<td>13</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul14">14</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul15">15</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul16">16</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul17">17</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul18">18</a></td>
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<td>19</td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul20">20</a></td>
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jul21">21</a></td>
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<td>22</td>
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<td colspan="7"> August 2014 </td>
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<td colspan="7" align=center><i> Month 5 of Project </i></td>
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<td align=center>sun</td>
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<td align=center>mon</td>
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<td align=center>1</td>
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<td align=center>3</td>
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<td align=center>6</td>
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<td align=center>9</td>
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<td align=center>10</td>
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<td align=center>11</td>
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<td align=center>13</td>
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<td align=center>14</td>
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<td align=center>17</td>
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<td align=center>18</td>
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<td align=center>24</td>
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<td align=center>25</td>
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<td align=center>28</td>
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<td align=center>31</td>
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<td colspan="7"> September 2014 </td>
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<td colspan="7" align=center><i> Month 6 of Project </i></td>
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<td align=center>sun</td>
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<td align=center>1</td>
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<td align=center>7</td>
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<td align=center>11</td>
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<td align=center>14</td>
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<td align=center>18</td>
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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>28</td>
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<td colspan="7"> October 2014 </td>
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<td colspan="7" align=center><i> Month 7 of Project </i></td>
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<td align=center>sun</td>
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<td align=center>mon</td>
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<td align=center>tue</td>
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<td align=center>thu</td>
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<td align=center>1</td>
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<td align=center>2</td>
+
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<td align=center>3</td>
+
-
<td align=center>4</td>
+
-
</tr>
+
-
<tr>
+
-
<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>
+
-
<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>
+
-
<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>
+
-
</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>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div id="rightnb">
+
-
 
+
-
<div class="day">
+
-
<a id="apr"><h1>April Overview</h1></a>
+
-
<div class="obj">Objectives</div>
+
-
<div class="lab">
+
-
<p>This is what we were trying to accomplish in April. </p>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="apr7"><h2> April 7 </h2></a>
+
-
 
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano and Matthew Faw</div>
+
-
<div class="lab">
+
-
 
+
-
<p>PCR of Z4EV from pMN10</p>
+
-
<ul>
+
-
<li>new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM) </li>
+
-
<li>4 tubes with 0, 0.3, 0.6, 1 uL template </li>
+
-
<ul><li>iGEM 3-step protocol with 65C anneal, 20 sec extend</li></ul>
+
-
<li>Note: did not dilute template beforehand (do this in future)</li>
+
-
</ul>
+
-
 
+
-
<p>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)</p>
+
-
<ul>
+
-
<li>Expected band size 715 bp </li>
+
-
<li>Control (no template) showed no band, lane 4 produced faint band </li>
+
-
<li>Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) </li>
+
-
</ul>
+
-
 
+
-
<p>Next steps: </p>
+
-
<ul>
+
-
<li>Run on gel to confirm (4/8) </li>
+
-
<li>digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8) </li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
 
+
-
<div class="day">
+
-
<h2><a id="apr8"> April 8 </a></h2>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano</div>
+
-
<div class="lab">
+
-
 
+
-
<p> Agarose Gel with Z4EV PCR products </p>
+
-
<ul>
+
-
<li>PCR from 04/07/2014 of Z4EV from pMN10</li>
+
-
<li>Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel) </li>
+
-
<ul>
+
-
<li>Expected band size 715 bp </li>
+
-
<li>Control (no template) showed no band, lane 4 produced faint band </li>
+
-
<li>Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb) </li>
+
-
</ul>
+
-
</ul>
+
-
 
+
-
<p>PCR cleanup of Z4EV PCR products</p>
+
-
<ul>
+
-
<li>PCR from 04/07/2014 of Z4EV from pMN10 </li>
+
-
<li>Used only lanes 2 and 3 (successful from gel) </li>
+
-
<li>Concentration 32.4 ng/uL in 50 uL </li>
+
-
</ul>
+
-
 
+
-
<p> Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1</p>
+
-
<ul>
+
-
<li>Both with SpeI-HF and NcoI-HF in Cutsmart </li>
+
-
<li>PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction </li>
+
-
<li>dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction </li>
+
-
</ul>
+
-
 
+
-
<p> PCR cleanup of Z4EV PCR digest product</p>
+
-
<ul>
+
-
<li>04/08 digest of 04/07 PCR</li>
+
-
<li> Results: Final concentration negligible, no DNA</li>
+
-
</ul>
+
-
 
+
-
<p> Next steps: </p>
+
-
<ul>
+
-
<li>Z4EV PCR again from pMN10</li>
+
-
<li>Digest of PCR in SpeI/NcoI</li>
+
-
<li>Gel extraction of digested TDH3-dCas9-MxiI</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="apr10"><h2> April 10 </h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano</div>
+
-
<div class="lab">
+
-
 
+
-
<p> PCR of Z4EV from pMN10</p>
+
-
<ul>
+
-
<li>Repeat of PCR from 4/7/14</li>
+
-
<li>Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p</li>
+
-
<li>Diluted pMN10 template to 1 ng/uL before use</li>
+
-
<li>8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction</li>
+
-
<ul><li>iGEM 3-step protocol with 65C anneal, 20 sec extend</li></ul>
+
-
<li>Results (see 4/11): expected bands present, but concentrations too low</li>
+
-
</ul>
+
-
 
+
-
<p> Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest</p>
+
-
<ul>
+
-
<li>Digest performed 4/8 with SpeI/NcoI</li>
+
-
<li>Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)</li>
+
-
<li>Obtained consistent band sizes, used 400 mg gel in extraction <</li>
+
-
<li>Gel picture: <img src="https://static.igem.org/mediawiki/2014/3/3c/4-10-14gel.png"></li>
+
-
<li> Results: obtained 20 uL product at 55 ng/uL (froze for later use)</li>
+
-
</ul>
+
-
<!-- https://2014.igem.org/File:4-10-14gel.png or https://static.igem.org/mediawiki/2014/3/3c/4-10-14gel.png -->
+
-
 
+
-
<p> Next steps: </p>
+
-
<ul>
+
-
<li> Run gel of PCR</li>
+
-
<li> Clean up PCR</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div class="day">
+
-
<a id="apr11"><h2>April 11</h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl"> Matthew Farnitano and Matthew Faw</div>
+
-
<div class="lab">
+
-
 
+
-
<p> Agarose gel to analyze PCR of Z4EV from pMN10</p>
+
-
<ul>
+
-
<li>PCR from 4/10</li>
+
-
<li>Expected band size: 715 bp</li>
+
-
<li>Showed band at expected size in all seven non-control lanes</li>
+
-
<li>Intensities appear low, increase in higher lanes (higher template conc.)</li>
+
-
<li>Gel picture: <img src="https://static.igem.org/mediawiki/2014/c/cd/4-11-14gel.png"> </li>
+
-
<!-- https://2014.igem.org/File:4-11-14gel.png or https://static.igem.org/mediawiki/2014/c/cd/4-11-14gel.png -->
+
-
</ul>
+
-
 
+
-
<p>PCR cleanup of Z4EV from 4/10 PCR</p>
+
-
<ul>
+
-
<li>final concentration from 7 tubes: 25.8 ng/uL in 30 uL</li>
+
-
<li>Need to improve</li>
+
-
</ul>
+
-
 
+
-
<p> Next Steps:</p>
+
-
<ul>
+
-
<li>Redo PCR of Z4EV. Things to try:</li>
+
-
<ul>
+
-
<li>Use previous cleanup as a template</li>
+
-
<li>use 1 uL enzyme instead of 0.5</li>
+
-
<li>slightly longer extension time</li>
+
-
<li>higher template concentration</li>
+
-
</ul>
+
-
<li>PCR cleanup: Things to try:</li>
+
-
<ul>
+
-
<li>Run PB buffer flow-through back through filter during step 7</li>
+
-
<li>let PE buffer sit in column (covered) while ethanol evaporates</li>
+
-
<li>let elution buffer sit on DNA for 2-3 minutes before spin</li>
+
-
</ul>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div class="day">
+
-
<a id="apr13"><h2>April 13 - Unfinished</h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl"> Matthew Farnitano and Garima Tomar</div>
+
-
<div class="lab">
+
-
 
+
-
<p> PCR of Z4EV from pMN10</p>
+
-
<ul>
+
-
<li>Third attempt (previously 4/7, 4/10)</li>
+
-
<li>oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p</li>
+
-
<li>8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction</li>
+
-
<ul>
+
-
<li>Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction</li>
+
-
<li>iGEM 3-step protocol with 65C anneal, 1 min extension</li>
+
-
</ul>
+
-
<li>Changes from previous: more template, more polymerase, longer extension</li>
+
-
</ul>
+
-
 
+
-
<p> Results (Date): </p>
+
-
<ul>
+
-
<li>???</li>
+
-
</ul>
+
-
 
+
-
<p> Next steps: </p>
+
-
<ul>
+
-
<li>Run on gel</li>
+
-
<li>Clean up PCR with new notes (see 4/11), see if concentration is better </li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="apr14"><h2>April 14</h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano</div>
+
-
<div class="lab">
+
-
 
+
-
<p> Agarose gel of Z4EV PCR</p>
+
-
<ul>
+
-
<li>Z4EV from pMN10, 4/13 PCR</li>
+
-
<li>2-log ladder and 8 identical reaction tubes</li>
+
-
<li>Expected band size ~715 bp</li>
+
-
<ul>
+
-
<li>All lanes showed band 700-800 bp</li>
+
-
<li>band intensity much greater than previous attempts</li>
+
-
</ul>
+
-
<li>Unable to take normal picture: used dark room and iPhone camera to photograph</li>
+
-
 
+
-
</ul>
+
-
 
+
-
<p>PCR cleanup of Z4EV PCR from 4/13</p>
+
-
<ul>
+
-
<li>Qiagen miniprep steps 7-10</li>
+
-
<li>8 tubes into 1 final product</li>
+
-
<li>Modifications to protocol:</li>
+
-
<ul>
+
-
<li>ran PB buffer through twice</li>
+
-
<li>let column sit, covered, for 10-15 min after PE buffer wash/discard step</li>
+
-
<li>let water for elution sit on column for 3 min before spin</li>
+
-
</ul>
+
-
<li>Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use)</li>
+
-
</ul>
+
-
 
+
-
<p>Next Steps: </p>
+
-
<ul>
+
-
<li>Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI</li>
+
-
<li>ligation of Z4EV with dCas9 backbone (obtained 4/10)</li>
+
-
<li>transformation</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="apr15"><h2>April 15</h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano</div>
+
-
<div class="lab">
+
-
 
+
-
<p> Restriction Digest of Z4EV-PCR with SpeI/NcoI</p>
+
-
<ul>
+
-
<li>PCR from 4/13, cleaned 4/14</li>
+
-
<li>Cut with SpeI-HF and NcoI-HF in Cutsmart buffer</li>
+
-
<ul>
+
-
<li>30 uL PCR product (407.5 ng/uL) in 50 uL reaction</li>
+
-
<li>Incubated at 37C for 4.5 hrs</li>
+
-
</ul>
+
-
</ul>
+
-
 
+
-
<p> PCR Cleanup of Restriction Digest of Z4EV</p>
+
-
<ul>
+
-
<li>Qiagen kit with modified methods described on 4/14 </li>
+
-
<li>Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop) </li>
+
-
</ul>
+
-
 
+
-
<p> Ligation of Z4EV PCR and dCas9-MxiI backbone</p>
+
-
<ul>
+
-
<li>Z4EV from pMN10, digested with NcoI and SpeI (4/15)</li>
+
-
<li>dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10</li>
+
-
<li>3:1 molar ratio backbone:insert</li>
+
-
<ul>
+
-
<li>20 uL total reaction - 15.3 uL backbone (55 ng/uL), 0.7 uL insert (248.1 ng/uL)</li>
+
-
</ul>
+
-
<li>Left in cold room overnight in 16C heat block</li>
+
-
</ul>
+
-
 
+
-
<p>Next steps: </p>
+
-
<ul>
+
-
<li>Transform ligated plasmid into E. coli, clone, then miniprep.</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="apr16"><h2>April 16</h2></a>
+
-
<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
+
-
<div class="ppl">Matthew Farnitano and Charlie Cooper</div>
+
-
<div class="lab">
+
-
<p> Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli</p>
+
-
<ul>
+
-
<li>Ligation left overnight for 22 hrs</li>
+
-
<li>Heat shock protocol with chemically competent E. coli</li>
+
-
<ul>
+
-
<li>1 sample plus 1 no-insert control</li>
+
-
<li>grown in SOC medium, plated on LB+Amp plates</li>
+
-
</ul>
+
-
<li> Results (Charlie Cooper) (4/17): No colonies on either plate</li>
+
-
<ul>
+
-
<li>Possibly used ineffective cells</li>
+
-
</ul>
+
-
</ul>
+
-
 
+
-
<p> Next steps: </p>
+
-
<ul>
+
-
<li>Digest and gel extraction of backbone</li>
+
-
<li>Ligation and Transformation</li>
+
-
</ul>
+
-
 
+
-
</div>
+
-
</div>
+
-
 
+
-
<div class="day">
+
-
<a id="apr"><h1>May, June Overview</h1></a>
+
-
<div class="obj">Objectives</div>
+
-
<div class="lab">
+
-
<p>This is what we were trying to accomplish in May and June. </p>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div class="day">
+
-
<a id="may29"><h2>May 29</h2></a>
+
-
<div class="obj">Objective: Prepare Chemically competent E. Coli</div>
+
-
<div class="ppl">Matthew Faw</div>
+
-
<div class="lab">
+
-
<p> Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli </p>
+
-
<ul>
+
-
<li> 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</li>
+
-
</ul>
+
-
<p>Next Steps: </p>
+
-
<ul>
+
-
<li> Transformation </li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
<div class="day">
+
-
<a id="jun1"><h2>June 1</h2></a>
+
-
<div class="obj">Objective: Transform Chemically competent E. Coli (CCEC)</div>
+
-
<div class="ppl">Matthew Faw</div>
+
-
<div class="lab">
+
-
<p> Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA</p>
+
-
<ul>
+
-
<li>Followed Charlie’s Cloning Protocols</li>
+
-
<ul>
+
-
<li>Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful</li>
+
-
</ul>
+
-
<li>Plated the transformed DNA and put in incubator at 37C</li>
+
-
<li>Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw</li>
+
-
</ul>
+
-
 
+
-
<p> Next Steps:</p>
+
-
<ul>
+
-
<li>Look at plates, compare cultures</li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div class="day">
+
-
<a id="jun2"><h2>June 2</h2></a>
+
-
<div class="obj">Objective: Redo yesterday’s transformation</div>
+
-
<div class="ppl">Matthew Faw</div>
+
-
<div class="lab">
+
-
<p> Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.</p>
+
-
<ul>
+
-
<li>Followed Charlie’s Cloning Protocols</li>
+
-
<ul><li>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</li></ul>
+
-
<li>Plated the transformed DNA on 6 separate plates, put in 37C overnight</li>
+
-
<li>Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol</li>
+
-
</ul>
+
-
 
+
-
<p>Next Steps:</p>
+
-
<ul>
+
-
<li>Look at plates, compare cultures, etc. </li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
-
 
+
-
 
+
-
 
+
-
6/4/14
+
-
 
+
-
Objective: Prepare buffers and mediums for new CCEC protocol
+
-
Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper
+
-
Prepare SOB Medium for bacterial transformation
+
-
Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
+
-
Made 1 L, autoclaved and stored in two 500 mL containers in cold room
+
-
medium still appeared cloudy before autoclave--may just be new recipe
+
-
Prepare CCMB 80 Buffer for making chemically competent E. coli cells
+
-
Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
+
-
Made 1 L, filtered and stored in two 500 mL containers in cold room
+
-
pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed
+
-
Autoclave two 500 mL culture flasks
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For CCEC protocol
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With water inside to remove detergent residues
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Next steps:
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Prepare CCEC
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Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC
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Matthew Faw
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The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed.  Today, we are trying to see if we can get any transformed cells to grow in plates. 
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Followed Charlie’s Cloning protocols,with slight modifications
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Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure
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Plated the transformed DNA on 2 separate plates, put in put in 37C overnight
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Results: No colonies grew (6/5/14)
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Next Steps:
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-Examine plates to see if any cultures grew
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-Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here: http://parts.igem.org/Help:Protocols/Competent_Cells
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-Lab currently in the process of making these CCEC
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Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped
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Matthew Faw, Charlie Cooper
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Prepare pdCas9 and pCsy4 to be miniprepped tomorrow
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Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol
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Put pCsy4 in a culture tube with 5ml SOC+Amp
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Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them
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Next steps:
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Miniprep plasmid DNA
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Latest revision as of 02:14, 18 October 2014