Team:UCL/Labbook

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<p class="major_title">Lab Weeks</p>
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<p class="major_title">Bacterial</p>
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    <p style="text-align: center;"> <a href="https://2013.igem.org/Team:UCL/Labbook/BacterialLabs/Week1" target="_blank"> Week1</a> | Week 2 | Week 3 | Week 4 | Week 5 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20</p>
 
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a>
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Click the links above to view the corresponding Lab diary weeks
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<p class="major_title">Mammalian</p>
 
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<!-- END CONTENT ------------------------------------------------------------------------------------------------------>
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    <p style="text-align: center;"> <a href="https://2013.igem.org/Team:UCL/Labbook/Week1" target="_blank"> Week1</a> | Week 2 | Week 3 | Week 4 | Week 5 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20</p>
 
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<script type="text/javascript" src="https://2013.igem.org/Team:UCL/static/footer.js?action=raw&ctype=text/javascript">
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<p class="major_title">June</p>
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</body>
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<p class="minor_title">Week 1-3</p>
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<p class="body_text">
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No lab work
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<p class="minor_title">Week 4</p>
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<p class="body_text">
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26th June - We are given safety training in the Advanced Centre for Biochemical Engineering in all relevant laboratories, as well as general procedures in case of emergency.
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</p>
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<p class="major_title">July</p>
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<p class="minor_title">Week 5-6</p>
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<p style="text-align: center;">
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No lab work
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</p>
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<p class="minor_title">Week 7</p>
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<p class="body_text">
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<b>Bacterial Lab</b>
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</p>
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<p class="body_text">
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15th July - The team is introduced to the laboratories which will be used during the summer for both bacterial and mammalian experiments.
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</p>
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<p class="body_text">
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16th July - <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> 5X M9 salts</a>, <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> minimal agar</a>,  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> 1.4% molten agar solution</a> and <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> 0.1M CaCl2/15% glycerol</a> were prepared for the generation of competent cells. Minimal agar plates were poured and <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> with W3110 Escherichia coli cells and left overnight to incubate at 37C.
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</p>
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<p class="body_text">
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17th July - Very little colony growth was observed from W3110 ''E.coli'' streaked plates. Plates were therefore left to incubate for a further 17 hours.
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</p>
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<p class="body_text">
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18th July - Sufficient colony growth allowed for the selection of a single colony from each plate. This was then inoculated in 5ul  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> LB media</a> + 100ul 1M MgSO4 and left to incu-shake overnight at 37C.
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</p>
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<p class="body_text">
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19th July - Cultures re-suspended in new <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> LB media</a> and 100µl aliquots placed into individual eppendorf tubes for placement at -80C.
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</p>
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<p class="body_text">
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<b>Mammalian Lab</b>
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</p>
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<p class="body_text">
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17th July -  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Mammalian cell culture and maintenance</a> training by Mrs. Ludmilla Ruban. Passaged primary MEF (mouse embryonic fibroblast) cells. Passage 3.
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</p>
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<p class="body_text">
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18th July - MEF passage 4
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</p>
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<p class="body_text">
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19th July - MEF passage 5
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</p>
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</p>
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<p class="minor_title">Week 8</p>
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<p class="body_text">
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<b>Bacterial Lab</b>
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</p>
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<p class="body_text">
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22nd July -<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Transformation</a> of our <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> with plasmid YB3110 was carried out.
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</p>
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<p class="body_text">
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23rd July - No colony growth was observed on  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Ampicillin plates</a> indicating no plasmid uptake. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation</a> was repeated with YB3110.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Vial</th>
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<th>Ampicillin Plate</th>
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<th>Plasmid Insertion</th>
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<th>Colony Count</th>
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</tr>
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<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>0</td>
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</tr>
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<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>0</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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24th July - No colony growth from main experiment indicating no plasmid uptake. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Transformation</a> was repeated.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Vial</th>
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<th>Ampicillin Plate</th>
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<th>Plasmid Insertion</th>
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<th>Colony Count</th>
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</tr>
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<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>0</td>
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</tr>
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<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>0</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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25th July - No colony growth and hence no plasmid uptake. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Transformation</a> was repeated.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Vial</th>
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-
<th>Ampicillin Plate</th>
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<th>Plasmid Insertion</th>
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<th>Colony Count</th>
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</tr>
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<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>0</td>
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</tr>
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<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>0</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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26th July - Once again our cells were unsuccessful in taking up the YB3110 plasmid. We concluded that our batch of competent cells were not competent and so these will not be used in any further experiments. A new batch of <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> are to be generated.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Vial</th>
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-
<th>Ampicillin Plate</th>
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<th>Plasmid Insertion</th>
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<th>Colony Count</th>
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</tr>
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<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>0</td>
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</tr>
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<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>0</td>
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</tr>
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</table>
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</p>
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<b>Mammalian Lab</b>
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</p>
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<p class="body_text">
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22nd July - MEF passage 6. Training ends.
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</p>
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<p class="minor_title">Week 9</p>
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<p class="body_text">
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<b>Bacterial Lab</b>
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</p>
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<p class="body_text">
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29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.
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</p>
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<p class="body_text">
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30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> miniprep</a>. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Vial</th>
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<th>Ampicillin Plate</th>
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-
<th>Plasmid Insertion</th>
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<th>Colony Count</th>
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</tr>
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<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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<td>Yes</td>
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<td>100+</td>
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</tr>
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<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>0</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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31st July - A new stock of <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> were generated and stored, these were tested for competence via <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation</a> using pSecTag2A and <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaking</a> onto <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> amp plates</a> -> incubate 37C o/n.
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</p>
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<p class="body_text">
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<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Ampicillin</a> was produced and stored. Following  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> miniprep</a> of pSecTag2A a <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> gel</a> was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A].  <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> 50X TAE diluted to 1X</a>.
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Item</th>
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<th>Volume (ul)</th>
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</tr>
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<tr>
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<td>DNA pSecTag2A</td>
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<td>5</td>
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</tr>
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<tr>
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<tr>
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<td>HindIII</td>
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<td>1</td>
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</tr>
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<tr>
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<td>Buffer</td>
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<td>1</td>
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</tr>
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<tr>
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<td>BSA</td>
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<td>0.5</td>
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</tr>
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<tr>
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<td>dH20</td>
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<td>2.5</td>
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</tr>
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<tr>
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<th>Total</th>
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<td>10</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.
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</p>
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</p>
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<p class="major_title">August</p>
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</p>
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<p class="body_text">
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1st August - Results from newly generated <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells transformed</a> with pSecTag2A:
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</p>
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<p class="body_text">
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-
 
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<table>
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-
<tr>
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-
<th>Vial</th>
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-
<th>Ampicillin Plate</th>
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-
<th>Plasmid Insertion</th>
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-
<th>Colony Count</th>
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-
</tr>
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-
<tr>
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<td>1 (Main)</td>
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<td>Yes</td>
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<td>Yes</td>
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<td>100+</td>
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-
</tr>
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-
<tr>
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<tr>
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<td>2 (Positive Control)</td>
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<td>No</td>
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-
<td>Yes</td>
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<td>100+</td>
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-
</tr>
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-
<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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<td>No</td>
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<td>10</td>
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</tr>
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</table>
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-
 
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</p>
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<p class="body_text">
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This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation protocol</a> from yesterday was repeated to see whether the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicilin</a> was working -> plates left for incubation 37C o/n.
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</p>
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<p class="body_text">
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<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Transformation</a> of <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.
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</p>
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<p class="body_text">
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2nd August - Plates displayed significant colony growth on <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> amp plates</a> indicating successful <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation</a>. Negative control however displayed slight colony growth - ampicillin not working effectively, new <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicillin</a> was therefore prepared. End of week inventory was recorded and stocks were topped up.
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</p>
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<p class="body_text">
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-
<table>
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-
<tr>
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-
<th>Vial</th>
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-
<th>Ampicillin Plate</th>
+
-
<th>Plasmid Insertion</th>
+
-
<th>Colony Count</th>
+
-
</tr>
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-
<tr>
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-
<td>1 (Main)</td>
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-
<td>Yes</td>
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-
<td>Yes</td>
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-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
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<td>2 (Positive Control)</td>
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-
<td>No</td>
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-
<td>Yes</td>
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<td>100+</td>
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-
</tr>
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-
<tr>
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<td>3 (Negative Control)</td>
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<td>Yes</td>
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-
<td>No</td>
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-
<td>15</td>
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-
</tr>
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-
</table>
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-
 
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-
</p>
+
-
<b>Mammalian Lab</b>
+
-
</p>
+
-
<p class="body_text">
+
-
29th July - Thawed and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu
+
-
</p>
+
-
<p class="body_text">
+
-
30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend
+
-
</p>
+
-
 
+
-
<p class="minor_title">Week 10</p>
+
-
<p class="body_text">
+
-
<b>Bacterial Lab</b>
+
-
</p>
+
-
<p class="body_text">
+
-
5th August - The new batch of <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicillin</a> was tested via <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation</a> of <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> with pSecTag2A. Plates were <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> spread</a> and incubated 37C o/n.
+
-
</p>
+
-
<p class="body_text">
+
-
6th August - Results from yesterday’s transformation:
+
-
</p>
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Vial</th>
+
-
<th>Ampicillin Plate</th>
+
-
<th>Plasmid Insertion</th>
+
-
<th>Colony Count</th>
+
-
</tr>
+
-
<tr>
+
-
<td>1 (Main)</td>
+
-
<td>Yes</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>2 (Positive Control)</td>
+
-
<td>No</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<td>3 (Negative Control)</td>
+
-
<td>Yes</td>
+
-
<td>No</td>
+
-
<td>15</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
This indicates that there is still an issue with the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicillin</a>, possibly a problem with the stock powder used. A final test with both old and new <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicillin</a> was carried out and compared without plasmid insertion.
+
-
</p>
+
-
<p class="body_text">
+
-
7th August -
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Vial</th>
+
-
<th>Ampicillin Plate</th>
+
-
<th>Plasmid Insertion</th>
+
-
<th>Colony Count</th>
+
-
</tr>
+
-
<tr>
+
-
<td>1 Old Ampicillin</td>
+
-
<td>Yes</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>2 New Ampicillin v2</td>
+
-
<td>No</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<td>3 Positive Control</td>
+
-
<td>Yes</td>
+
-
<td>No</td>
+
-
<td>15</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
Results indicated that the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> ampicillin</a> source may not have been fully functional, therefore a new source of amp powder was located and amp was remade.
+
-
</p>
+
-
<p class="body_text">
+
-
8th August - Preparation of 4x <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Amp plates</a> and 4x <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> no drug plates</a> for storage in the fridge.
+
-
</p>
+
-
<p class="body_text">
+
-
9th August - Meeting with Darren Nesbeth, requirements for upcoming weeks: create <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a> of both pSecTag2A and pSB1C3, purify and extract pure DNA for pSecTag2A and pSB1C3.
+
-
</p>
+
-
</p>
+
-
<b>Mammalian Lab</b>
+
-
</p>
+
-
<p class="body_text">
+
-
6th August - Checked HeLa cells. Cells are growing slow, left to grow for a few more days
+
-
</p>
+
-
<p class="body_text">
+
-
8th August - HeLa cells are about 30% confluent. Changed media.
+
-
</p>
+
-
 
+
-
<p class="minor_title">Week 11</p>
+
-
<p class="body_text">
+
-
<b>Bacterial Lab</b>
+
-
</p>
+
-
<p class="body_text">
+
-
12th August - <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a> was produced and stored at -20C.
+
-
In order to produce <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a>, pSecTag2A and <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37C o/n.
+
-
</p>
+
-
<p class="body_text">
+
-
13th August - Results from the following plates:
+
-
</p>
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Vial</th>
+
-
<th>Ampicillin Plate</th>
+
-
<th>Plasmid Insertion</th>
+
-
<th>Colony Count</th>
+
-
</tr>
+
-
<tr>
+
-
<td>pSecTag2A Cells</td>
+
-
<td>Yes</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>W3110 Cells Amp</td>
+
-
<td>Yes</td>
+
-
<td>No</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>pSecTag +ve control</td>
+
-
<td>No</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PSB1C3 Cells</td>
+
-
<td>Yes</td>
+
-
<td>Yes</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>W3110 Cells Chlor</td>
+
-
<td>Yes</td>
+
-
<td>No</td>
+
-
<td>25</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PSB1C3 +ve control</td>
+
-
<td>No</td>
+
-
<td>Yes</td>
+
-
<td>0</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
This indicated that the pSecTag2A cells are acceptable to use for <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stock</a> generation and also for plasmid purification. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Miniprep</a> was performed on the two incubated Falcon tubes from yesterday.
+
-
</p>
+
-
<p class="body_text">
+
-
The results concerning <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> indicated that the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> chloramphenicol</a> did not work, and the glycerol stock is dead. Therefore a new <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stock</a> was sought after and <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> chloramphenicol</a> was remade.
+
-
Amp & Cmp were remade and pSB1C3 was located in the 2012 distribution kit. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Amp & Cmp</a> were both tested by producing 1x positive and 1x negative plate for each antibiotic <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> with W3110 cells ->left to incubate o/n at 37C.
+
-
<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a> of pSecTag2A was grown, and a <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> miniprep</a> was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.
+
-
</p>
+
-
<p class="body_text">
+
-
14th August - Two analytical digests of pSecTag2A with EcoR1-HF and Dpn1 were carried out.
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Item A</th>
+
-
<th>Volume A (ul)</th>
+
-
<th>Item B</th>
+
-
<th>Volume B (ul)</th>
+
-
</tr>
+
-
<tr>
+
-
<td>pSecTag2A</td>
+
-
<td>5</td>
+
-
<td>pSecTag2A</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>EcoR1-HF</td>
+
-
<td>1</td>
+
-
<td>Dpn1</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Buffer 4</td>
+
-
<td>1</td>
+
-
<td>Buffer 4</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>BSA</td>
+
-
<td>0.5</td>
+
-
<td>BSA</td>
+
-
<td>0.5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>dH20</td>
+
-
<td>2.5</td>
+
-
<td>dH20</td>
+
-
<td>2.5</td>
+
-
</tr>
+
-
<tr>
+
-
<th>Total</th>
+
-
<td>10</td>
+
-
<th>Total</th>
+
-
<td>10</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
[insert image of gel]
+
-
</p>
+
-
[link to enzyme conditions]
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
The <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> glyc stock (from HQ) was from the 2012 iGEM box in MMP -20C
+
-
</p>
+
-
<p class="body_text">
+
-
Told to use a ratio of 3:1 inoculum:80% glycerol when making glyc stocks
+
-
</p>
+
-
<p class="body_text">
+
-
Darren's instructions:
+
-
</p>
+
-
<p class="body_text">
+
-
<b>pSB1C3</b>
+
-
</p>
+
-
<p class="body_text">
+
-
Transfer entire glycerol tube contents to 10mL LB ND in a 50mL Falcon & measure OD. Place in 37C shakee overnight. Measure OD next morning. If there is growth in the inoculum use a loop to streak onto cmp and ND plates again.
+
-
</p>
+
-
<p class="body_text">
+
-
<b>pSecTag2A</b>
+
-
</p>
+
-
<p class="body_text">
+
-
50ul of 2013 pSecTag2A glyc stock to inoculate 10mL LB Amp in a 50mL Falcon. Grow overnight and the next day generate 15x glyc stocks sing 1.5mL eppendorfs -> store in MMP -20C.
+
-
</p>
+
-
<p class="body_text">
+
-
15th August - OD results for pSB1C3 LB ND inoculum
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Plasmid</th>
+
-
<th>OD Before</th>
+
-
<th>OD After</th>
+
-
</tr>
+
-
<tr>
+
-
<td>PSB1C3 (LB ND)</td>
+
-
<td>0.052</td>
+
-
<td>0.5</td>
+
-
</tr>
+
-
<table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
15x eppendorfs of pSecTag2A Amp glycerol stock and 4x eppendorfs of <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> ND <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stock</a> was prepared.
+
-
pSB1C3 LB ND was <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto two plates (1x Cmp, 1xND) ->incubate o/n @37C
+
-
Remaining 2ml of pSB1C3 LB ND (from falcon) is used to purify and conduct an analytical digest (16th August).
+
-
</p>
+
-
<p class="body_text">
+
-
Restriction digest of pSecTag2A:
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>ul</th>
+
-
<th>EcoR1 single digest</th>
+
-
<th>Spe1 single digest</th>
+
-
<th>Double digest</th>
+
-
<th>Uncut</th>
+
-
</tr>
+
-
<tr>
+
-
<td>pSecTag2A</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>EcoR1</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Spe1</td>
+
-
<td>0</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>BSA</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Buffer 4</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>dH20</td>
+
-
<td>2.5</td>
+
-
<td>2.5</td>
+
-
<td>1.5</td>
+
-
<td>3.5</td>
+
-
</tr>
+
-
<tr>
+
-
<th>Total</th>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
[insert image of gel]
+
-
</p>
+
-
<p class="body_text">
+
-
iRRE+PC+RBS and PC+RBS (containing <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>, taken from 2012 iGEM boxes) were plated onto <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> cmp</a> plates -> incubated o/n @37C
+
-
</p>
+
-
<p class="body_text">
+
-
Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml cmp -> incushaker o/n
+
-
</p>
+
-
<p class="body_text">
+
-
16th August - Falcons were retrieved:
+
-
</p>
+
-
<p class="body_text">
+
-
RESULTS
+
-
</p>
+
-
<p class="body_text">
+
-
Inoculum:     
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Falcon contents (+LB+Amp) ul</th>
+
-
<th>Colony growth</th>
+
-
<th>Absorbance</th>
+
-
</tr>
+
-
<tr>
+
-
<td>IRRE+PC+RBS (15ul)</td>
+
-
<td>Yes</td>
+
-
<td>0.8</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>IRRE+PC+RBS (5ul)</td>
+
-
<td>Yes</td>
+
-
<td>0.7</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PC+RBS (15ul)/td>
+
-
<td>No</td>
+
-
<td>0.06</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PC+RBS (5ul)</td>
+
-
<td>No</td>
+
-
<td>0.08</td>
+
-
<tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
Plates
+
-
</p>
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Plate contents (+LB+Amp) ul</th>
+
-
<th>Colony growth</th>
+
-
<th>Absorbance</th>
+
-
</tr>
+
-
<tr>
+
-
<td>IRRE+PC+RBS</td>
+
-
<td>Yes</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>IRRE+PC+RBS</td>
+
-
<td>No</td>
+
-
<td>100+</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PC+RBS/td>
+
-
<td>Yes</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>PC+RBS</td>
+
-
<td>No</td>
+
-
<td>20</td>
+
-
<tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
The PC+RBS plates & falcons were discarded
+
-
</p>
+
-
<p class="body_text">
+
-
IRRE+PC+RBS (15ul inoculum) -> underwent <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> miniprep</a>
+
-
</p>
+
-
<p class="body_text">
+
-
IRRE+PC+RBS (5ul inoculum) -> 4x <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerols</a> were made: 500ul culture + 166ul 80% glycerol, stored in MMP -20C iGEM 2013 box.
+
-
</p>
+
-
<p class="body_text">
+
-
pSB1C3 Gel sd: EcoR1 & Pst1 + dd
+
-
</p>
+
-
 
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Item (ul)</th>
+
-
<th>EcoR1</th>
+
-
<th>Pst1</th>
+
-
<th>Double digest</th>
+
-
<th>Uncut</th>
+
-
</tr>
+
-
<tr>
+
-
<td>pSB1C3</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>EcoR1</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Pst1</td>
+
-
<td>0</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Buffer 3</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>BSA</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
<td>0.5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>dH20</td>
+
-
<td>2.5</td>
+
-
<td>2.5</td>
+
-
<td>1.5</td>
+
-
<td>4.5</td>
+
-
</tr>
+
-
<tr>
+
-
<th>Total</th>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
<td>10</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
3ul loading dye to each solution
+
-
</p>
+
-
<p class="body_text">
+
-
Lanes:
+
-
</p>
+
-
<p class="body_text">
+
-
2 - Hyperladder, 4 - EcoRI, 5 - PstI, 6 - dd, 7 - Uncut
+
-
</p>
+
-
<p class="body_text">
+
-
Results from gel: 1kb DNA ladder showed on gel. No bands for EcoR1, Pst1, DD and Uncut plasmid were seen - indicating that no DNA was present -> possibly due to problems with the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> miniprep</a>.
+
-
</p>
+
-
<p class="body_text">
+
-
Therefore from the <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> iGEM 2012 plate 5</a> (distribution kit) well 23.O, DNA containing BBa_j04450 (colonies are clearly red in colour - RFP) in pSB1C3 was located. The dried DNA was <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> resuspended</a> in 10ul dH2O, left to sit for 5 minutes, then <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformed</a> into <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> competent cells</a> -> <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto plates and left to incubate o/n @30C over the weekend.
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
<b>Mammalian Lab</b>
+
-
</p>
+
-
<p class="body_text">
+
-
12th August - HeLa cells have confluency of about 60%. Passaged the HeLa cells and ‘backup’ HeLa cells. We set up two 6-well plates  at 0.25 x 10^6 cells/ml in preparation for Zeocin kill curve. Cell count involved (0.45 cells/ml).
+
-
</p>
+
-
<p class="body_text">
+
-
13th August -
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
Prepared media with various concentrations of zeocin for each of 6 wells.
+
-
</p>
+
-
 
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
<tr>
+
-
<th>Concentration of zeocin (µg/ml)</th>
+
-
<th>Volume of zeocin (ml)</th>
+
-
<th>Volume of DMEM + 10% FBS + 2 mM L-Glu (ml)</th>
+
-
</tr>
+
-
<tr>
+
-
<td>0</td>
+
-
<td>0</td>
+
-
<td>30.0</td>
+
-
</tr>
+
-
<tr>
+
-
<tr>
+
-
<td>50</td>
+
-
<td>15</td>
+
-
<td>30.0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>100</td>
+
-
<td>30</td>
+
-
<td>30.0</td>
+
-
</tr>
+
-
<tr>
+
-
<td>250</td>
+
-
<td>75</td>
+
-
<td>29.9</td>
+
-
</tr>
+
-
<tr>
+
-
<td>500</td>
+
-
<td>150</td>
+
-
<td>29.9</td>
+
-
</tr>
+
-
<tr>
+
-
<td>1000</td>
+
-
<td>300</td>
+
-
<td>29.7</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
T75 flasks: - 90% confluency
+
-
T25 flasks - 30% confluency
+
-
6-well plates - 30% confluency
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
14th August -  T25 flasks 90% confluency
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
Disc 1                                                Disc 2
+
-
</p>
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
</tr>
+
-
<th></th>
+
-
<th></th>
+
-
<th>Disc 1</th>
+
-
<th></th>
+
-
<th></th>
+
-
<th>Disc 2</th>
+
-
<th></th>
+
-
</tr>
+
-
</tr>
+
-
<th>Concentration of zeocin (µg/ml)</th>
+
-
<th>Confluency (%)</th>
+
-
<th>Cell Appearance</th>
+
-
<th>Comment</th>
+
-
<th>Confluency (%)</th>
+
-
<th>Cell Appearance</th>
+
-
<th>Comment</th>
+
-
</tr>
+
-
<tr>
+
-
<td>0</td>
+
-
<td>65</td>
+
-
<td>Healthy</td>
+
-
<td>Healthy, few swell</td>
+
-
<td>65</td>
+
-
<td>Healthy</td>
+
-
<td>minimal floaters</td>
+
-
</tr>
+
-
<tr>
+
-
<td>50</td>
+
-
<td>40</td>
+
-
<td>Healthy, few swell</td>
+
-
<td>few floaters</td>
+
-
<td>65</td>
+
-
<td>occasional swell</td>
+
-
<td>moderate floaters</td>
+
-
</tr>
+
-
<tr>
+
-
<td>100</td>
+
-
<td>40</td>
+
-
<td>Half/moderate swell</td>
+
-
<td>Moderate floaters, minor infection</td>
+
-
<td>50</td>
+
-
<td>40% swelling</td>
+
-
<td>minimal floaters</td>
+
-
</tr>
+
-
<tr>
+
-
<td>250</td>
+
-
<td>25</td>
+
-
<td>most/moderate swell</td>
+
-
<td>many floaters</td>
+
-
<td>60</td>
+
-
<td>moderate/severe swelling</td>
+
-
<td>many floaters</td>
+
-
</tr>
+
-
<tr>
+
-
<td>500</td>
+
-
<td>40</td>
+
-
<td>most clumps dead/ swell</td>
+
-
<td>many floaters, possibly infection</td>
+
-
<td>65</td>
+
-
<td>severe swelling all over</td>
+
-
<td>large number of floaters</td>
+
-
</tr>
+
-
<tr>
+
-
<td>1000</td>
+
-
<td>45</td>
+
-
<td>very severe death/ swell</td>
+
-
<td>many floaters, minor infection</td>
+
-
<td>60</td>
+
-
<td>severe swelling all over</td>
+
-
<td>moderate number of floaters</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
The two T25 flasks with revived HeLa have confluency of about 90%; T75 flask with ‘backup’ cells 70% confluent. The T25 flasks are discarded.
+
-
 
+
-
</p>
+
-
<p class="body_text">
+
-
16th August -
+
-
 
+
-
<p class="body_text">
+
-
<table>
+
-
</tr>
+
-
<th></th>
+
-
<th></th>
+
-
<th>Disc 1</th>
+
-
<th></th>
+
-
<th></th>
+
-
<th>Disc 2</th>
+
-
<th></th>
+
-
</tr>
+
-
</tr>
+
-
<th>Concentration of zeocin (µg/ml)</th>
+
-
<th>Confluency (%)</th>
+
-
<th>Cell Appearance</th>
+
-
<th>Floaters</th>
+
-
<th>Confluency (%)</th>
+
-
<th>Cell Appearance</th>
+
-
<th>Floaters</th>
+
-
</tr>
+
-
<tr>
+
-
<td>0</td>
+
-
<td>90</td>
+
-
<td>Healthy</td>
+
-
<td>Minimal</td>
+
-
<td>95</td>
+
-
<td>Healthy</td>
+
-
<td>minimal</td>
+
-
</tr>
+
-
<tr>
+
-
<td>50</td>
+
-
<td>80</td>
+
-
<td>minor swell</td>
+
-
<td>moderate</td>
+
-
<td>90</td>
+
-
<td>minor swell</td>
+
-
<td>moderate</td>
+
-
</tr>
+
-
<tr>
+
-
<td>100</td>
+
-
<td>65</td>
+
-
<td>Minor swell, minor death</td>
+
-
<td>Many</td>
+
-
<td>75</
+

Latest revision as of 00:25, 5 October 2013

Lab Weeks

Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18

Click the links above to view the corresponding Lab diary weeks

Retrieved from "http://2013.igem.org/Team:UCL/Labbook"