Team:Lethbridge Canada/notebook may

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<span id="title_first">Lethbridge</span>
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<span id="title_second">iGEM Team</span>
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<div id="mainContent">
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<div id="notebook_header">
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<div id="notebook_links">
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<h1 id="notebook_links_header">Notebook Links</h1>
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<ul id="notebook_march_links">
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<li class="notebook_link_title"><a href="notebook_march.html">March</a></li>
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<li><a href="notebook_march.html#entry_one">March 12, 2013: Transformation</a></li>
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<li><a href="notebook_march.html#entry_two">March 13, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="notebook_march.html#entry_three">March 13, 2013: Miniprep and Glycerol Stock</a></li>
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<li><a href="notebook_march.html#entry_four">March 15, 2013: Restriction Digestion</a></li>
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<li><a href="notebook_march.html#entry_five">March 16, 2013: Agarose Gel</a></li>
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<li><a href="notebook_march.html#entry_six">March 18, 2013: Agarose Gel</a></li>
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<li><a href="notebook_march.html#entry_seven">March 19, 2013: Transformation</a></li>
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<li><a href="notebook_march.html#entry_eight">March 20, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="notebook_march.html#entry_nine">March 21, 2013: Miniprep and Glycerol Stock</a></li>
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<li><a href="notebook_march.html#entry_ten">March 27, 2013: Transformation</a></li>
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<li><a href="notebook_march.html#entry_eleven">March 28, 2013: Transformation</a></li>
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<li><a href="notebook_march.html#entry_twelve">March 28, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="notebook_march.html#entry_thirteen">March 29, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="notebook_march.html#entry_fourteen">March 29, 2013: Picking Cells of -80 Glycerol Stock</a></li>
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</ul>
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<ul id="notebook_april_links">
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<li class="notebook_link_title"><a href="notebook_april.html">April</a></li>
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<li><a href="notebook_april.html#entry_one">April 2, 2013: Restriction - Ligation</a></li>
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<li><a href="notebook_april.html#entry_two">April 2, 2013: Ligation</a></li>
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<li><a href="notebook_april.html#entry_three">April 3, 2013: Transformation</a></li>
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<li><a href="notebook_april.html#entry_four">April 4, 2013: Re-Plating Transformation</a></li>
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<li><a href="notebook_april.html#entry_five">April 5, 2013: Miniprep of ON Cultures</a></li>
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<li><a href="notebook_april.html#entry_six">April 5, 2013: UV Spectroscopy DNA Concentrations</a></li>
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<li><a href="notebook_april.html#entry_seven">April 6, 2013: Miniprep From Ligations</a></li>
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<li><a href="notebook_april.html#entry_eight">April 7, 2013: PCR</a></li>
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<li><a href="notebook_april.html#entry_nine">April 8, 2013: Restriction Digestion of PCR Parts</a></li>
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<li><a href="notebook_april.html#entry_ten">April 8, 2013: 3% Agarose Gel of Digested PCR Parts</a></li>
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<li><a href="notebook_april.html#entry_eleven">April 8, 2013: Gel Extraction</a></li>
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<li><a href="notebook_april.html#entry_twelve">April 9, 2013: Repeat PCR</a></li>
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<li><a href="notebook_april.html#entry_thirteen">April 9, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="notebook_april.html#entry_fourteen">April 10, 2013: Miniprep</a></li>
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<li><a href="notebook_april.html#entry_fifteen">April 10, 2013: Restriction</a></li>
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<li><a href="notebook_april.html#entry_sixteen">April 10, 2013: Agarose Gel of PCR 1%</a></li>
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<li><a href="notebook_april.html#entry_seventeen">April 11, 2013: Miniprep</a></li>
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<li><a href="notebook_april.html#entry_eighteen">April 12, 2013: Restriction Digestion</a></li>
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<li><a href="notebook_april.html#entry_nineteen">April 12, 2013: PCR and Cell Reculturization</a></li>
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<li><a href="notebook_april.html#entry_twenty">April 12, 2013: Miniprep</a></li>
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<li><a href="notebook_april.html#entry_twenty_one">April 14, 2013: Miniprep</a></li>
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<li><a href="notebook_april.html#entry_twenty_two">April 14, 2013: 1% Agarose Gel</a></li>
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<li><a href="notebook_april.html#entry_twenty_six">April 15, 2013: Restriction/Digestion</a></li>
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<li><a href="notebook_april.html#entry_twenty_seven">April 15, 2013: Restriction/Digestion</a></li>
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<li><a href="notebook_april.html#entry_twenty_three">April 15, 2013: 1% Agarose Gel of Promoters</a></li>
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<li><a href="notebook_april.html#entry_twenty_four">April 17, 2013: Transformation</a></li>
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<li><a href="notebook_april.html#entry_twenty_five">April 30, 2013: 1% Agarose Gel of Promoter Constructs</a></li>
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</ul>
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<ul id="notebook_may_links">
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<li class="notebook_link_title"><a href="notebook_may.html">May</a></li>
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<li><a href="notebook_may.html#entry_one">May 1, 2013: Ligation</a></li>
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<li><a href="notebook_may.html#entry_two">May 4, 2013: Restriction Digest</a></li>
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<li><a href="notebook_may.html#entry_three">May 4, 2013: 1% Agarose Gel of Promoter Constructs</a></li>
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<li><a href="notebook_may.html#entry_four">May 4, 2013: Gel Extractions</a></li>
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<li><a href="notebook_may.html#entry_five">May 5, 2013: Transformation</a></li>
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<li><a href="notebook_may.html#entry_six">May 7, 2013: Restriction</a></li>
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<li><a href="notebook_may.html#entry_seven">May 7, 2013: Gel Extraction</a></li>
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<li><a href="notebook_may.html#entry_eight">May 7, 2013: Gel Extracted on Conformation Gel</a></li>
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<li><a href="notebook_may.html#entry_nine">May 8, 2013: Ligations</a></li>
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<li><a href="notebook_may.html#entry_ten">May 9, 2013: PCR of Ligation Mixture</a></li>
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<li><a href="notebook_may.html#entry_eleven">May 13, 2013: Restriction of PCR Ligations Gel Conformation</a></li>
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<li><a href="notebook_may.html#entry_twelve">May 14, 2013: Transformation of Ligations</a></li>
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<li><a href="notebook_may.html#entry_thirteen">May 15, 2013: Ligation</a></li>
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<li><a href="notebook_may.html#entry_fourteen">May 15, 2013: Picking Cells</a></li>
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<li><a href="notebook_may.html#entry_fifteen">May 16, 2013: Picking Cells</a></li>
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<li><a href="notebook_may.html#entry_sixteen">May 16, 2013: 10 Fold Miniprep</a></li>
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<li><a href="notebook_may.html#entry_seventeen">May 17, 2013: Agarose Gel</a></li>
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<li><a href="notebook_may.html#entry_eighteen">May 17, 2013: Miniprep</a></li>
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<li><a href="notebook_may.html#entry_nineteen">May 17, 2013: Glycerol Stock</a></li>
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</ul>
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</div>
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</div>
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<div id="notebook_body">
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<div class="notebook_entry">
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<div id="entry_one">
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<h1 class="notebook_entry_header">Ligations</h1>
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<ul class="notebook_entry_list">
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<li>Name: Yoyo Yao</li>
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<li>Date: May 1, 2013</li>
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<li class="notebook_nested_start">Ligation of Parts – Upstream, Ligated at sites S & P:
 +
<ul class="notebook_nested_list">
 +
<li>J23100</li>
 +
<li>J23113</li>
 +
<li>J23108 +</li>
 +
<li>J23108 –</li>
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<li>All promoters already attached to Destination Plasmid J61002</li>
 +
</ul>
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<li class="notebook_nested_start">Downstream, Ligated at sites X & P</li>
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<ul class="notebook_nested_list">
 +
<li>J23100</li>
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</ul>
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</li>
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</ul>
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<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
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<div class="notebook_entry">
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<div id="entry_two">
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<h1 class="notebook_entry_header">Restriction Digest</h1>
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<div class="restrict_width">
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<ul class="notebook_entry_list">
 +
<li>Name: Mackenzie</li>
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<li>Date: May 4, 2013</li>
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<li>35µL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1</li>
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</ul>
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</div>
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<ul>
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<li>
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<table class="table_two table_position">
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<thead>
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<tr>
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<th colspan="2">Master Mix</th>
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</tr>
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</thead>
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<tbody>
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<tr>
 +
<td>Milliq</td>
 +
<td>36µl</td>
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</tr>
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<tr>
 +
<td>BSA</td>
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<td>45µl</td>
 +
</tr>
 +
<tr>
 +
<td>10x buffer 2 NEB</td>
 +
<td>45µl</td>
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</tr>
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</tbody>
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</table>
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</li>
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</ul>
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<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
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<div class="notebook_entry">
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<div id="entry_three">
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<div class="restrict_width">
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<h1 class="notebook_entry_header">1% Agarose Gel of Promoter Constructs</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Mackenzie</li>
 +
<li>Date: May 4, 2013</li>
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</ul>
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</div>
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<ul>
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<li>
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<table class="table_three table_position">
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<thead>
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<tr>
 +
<th scope="col">Lane #</th>
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<th scope="col">Contents</th>
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<th scope="col">Volume</th>
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</tr>
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</thead>
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<tbody>
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<tr>
 +
<td>1</td>
 +
<td>1KB LADDER</td>
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<td>6µL</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>J23100_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>J23100_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>J23100_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>J23100_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>J23108_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<td>J23108_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<td>J23108_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>9</td>
 +
<td>J23108_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>J23113_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>11</td>
 +
<td>J23113_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>12</td>
 +
<td>J23113_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>13</td>
 +
<td>J23113_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>14</td>
 +
<td>J23114_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>15</td>
 +
<td>J23114_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>16</td>
 +
<td>J23114_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
<tr>
 +
<td>17</td>
 +
<td>J23114_J61002</td>
 +
<td>25µL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</li>
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</ul>
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<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
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<div class="notebook_entry">
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<div id="entry_four">
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<h1 class="notebook_entry_header">Gel Extraction</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: May 4, 2013</li>
 +
<li>J23108 J61002</li>
 +
<li>J23113 J61002</li>
 +
<li>J23114 J61002</li>
 +
<li>J23100 J61002</li>
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<li>Eluted 50 µL</li>
 +
</ul>
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<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
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<div class="notebook_entry">
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<div id="entry_five">
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<h1 class="notebook_entry_header">Transformation</h1>
 +
<div class="restrict_width_small">
 +
<ul class="notebook_entry_list">
 +
<li>Name: Mackenzie</li>
 +
<li>Date: May 5, 2013</li>
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</ul>
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</div>
 +
<ul>
 +
<li>
 +
<table class="table_four table_position">
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<thead>
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<tr>
 +
<th scope="col">Tube #</th>
 +
<th scope="col">Part #</th>
 +
<th scope="col">Plasmid</th>
 +
<th scope="col">Volume of Competent Cells</th>
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</tr>
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</thead>
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<tbody>
 +
<tr>
 +
<td>1</td>
 +
<td>J23100+J06702</td>
 +
<td>J61002</td>
 +
<td>20µL</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>J23108+J06702</td>
 +
<td>J61002</td>
 +
<td>20µL</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>J23113+J06702</td>
 +
<td>J61002</td>
 +
<td>20µL</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>J23108-J06702</td>
 +
<td>J61002</td>
 +
<td>20µL</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</li>
 +
</ul>
 +
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_six">
 +
<h1 class="notebook_entry_header">Restriction</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Chris Isaac</li>
 +
<li>Date: May 7, 2013</li>
 +
<li>Cut with ‘X’ and ‘P’</li>
 +
<li>J06702 – RBS-mcherry-DT</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_seven">
 +
<h1 class="notebook_entry_header">Gel Extraction</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Katie T. & Elaine B.</li>
 +
<li>Date: May 7, 2013</li>
 +
<li>J06702</li>
 +
<li>Eluted at 30µL</li>
 +
<li><img src="images/gel-extraction-05-07-2013.gif" alt="Gel Extraction Done on May 7, 2013" /></li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_eight">
 +
<div class="restrict_width">
 +
<h1 class="notebook_entry_header">Gel Extracted Parts on Conformation Gel</h1>
 +
<ul class="notebook_entry_list">
 +
<li>1µl of 1kb Ladder ng/0.5µg</li>
 +
<li>2µl DNA in all samples</li>
 +
<li class="move_up_table_two_long"><img src="images/gel-extracted-parts-on-conformation-gel.gif" alt="Gel Extracted Parts on Conformation Gel Image" /></li>
 +
</ul>
 +
</div>
 +
<ul
 +
<li>
 +
<table class="table_two table_position">
 +
<thead>
 +
<tr>
 +
<th>Lane #</th>
 +
<th>Content</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>1</td>
 +
<td>Ladder</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>J06702</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>J23113–J61002 mc05/04/13mdy</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>J23108–J61002 mc05/04/13</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>J23100–J61002 mc05/04/13</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>J23114–J61002 mc05/04/13</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<td>Ladder</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<td>J23100–J 61002 EK150413dmy</td>
 +
</tr>
 +
<tr>
 +
<td>9</td>
 +
<td>J23108–J61002 EK150413</td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>J23113–J61002EK150413</td>
 +
</tr>
 +
<tr>
 +
<td>11</td>
 +
<td>J23114 –J61002 EK150413</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
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</div>
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</div>
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 +
<div class="notebook_entry">
 +
<div id="entry_nine">
 +
<h1 class="notebook_entry_header">Gel Extraction</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Carissa K.</li>
 +
<li>Date: May 8, 2013</li>
 +
<li>2ul of J06702 mix with 4ul of each; J23100, J23108, J23113, J23114 all in plasmid J61002 (amp)</li>
 +
<li>In the Thermo-cycler at 16 degrees Celsius overnight  followed by heat kill.</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_ten">
 +
<h1 class="notebook_entry_header">PCR of Ligation Mixture(PCR to verify insert in clone vector)</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Chris Isaac</li>
 +
<li>Date: May 9, 2013</li>
 +
<li>X7 Master Mix, Taq polymerase</li>
 +
<li>J23100_J61002 (control)</li>
 +
<li>J06702_pSB1AK2 (control)</li>
 +
<li>J23100_J61002_J06702</li>
 +
<li>J23108_J601002_J06702</li>
 +
<li>J23113_J61002_J06702</li>
 +
<li>J23114_J61002_J06702</li>
 +
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_eleven">
 +
<h1 class="notebook_entry_header">Restriction of PCR Ligations Gel Conformation</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Patrick O'</li>
 +
<li>Date: May 13, 2013</li>
 +
<li>Restriction of May 9th pcr ligation at E and P and ran on 1.0% Gel (refreshed ETH Bromide)</li>
 +
<li>Ladder lane 2</li>
 +
<li>2space</li>
 +
<li>J06702_pSB1AK2 (control)</li>
 +
<li>2spaces</li>
 +
<li>J23100_J61002 (control)</li>
 +
<li>spaces</li>
 +
<li>J23100_J61002_J06702</li>
 +
<li>J23108_J601002_J06702</li>
 +
<li>J23113_J61002_J06702</li>
 +
<li>J23114_J61002_J06702</li>
 +
<li>Ladder</li>
 +
<li>Conclusion: Ligations were not Successful</li>
 +
<li><img src="images/gel-extraction-05-13-2013.gif" alt="Restriction of PCR Ligations Gel Conformation Image" /></li>
 +
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_twelve">
 +
<h1 class="notebook_entry_header">Transformation of Ligations</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: May 14, 2013</li>
 +
<li>Transformed ligation of J06702 with various promoters in plasmid J61002 : J23100, J23108, J23113, J23114 From May 8, 2013.</li>
 +
<li>Transformed 3ul ligation mix into 30ul of Dh5 alpha competent cells, used puc19 control. Heat shocked for 30second at 42 degrees Celsius added 200ul warm LB, incubated at 37 for 1 hour, Plated 100ul on amp LB Agar plates. Overnight at 37degrees Celsius.</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_thirteen">
 +
<h1 class="notebook_entry_header">Ligation Round 2 of May 14th Gel Extracted Parts</h1>
 +
<ul class="notebook_entry_list">
 +
<li>11ul dh20</li>
 +
<li>2ul t4 dna ligase buffer</li>
 +
<li>4ul DNA J06702</li>
 +
<li>2ul DNA J23100, J23108, J23113, J23114 in plasmid J61002</li>
 +
<li>1ul T4 dna Ligase</li>
 +
<li>Into the Thermo-cycler overnight at 16 degrees Celsius.</li>
 +
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_fourteen">
 +
<h1 class="notebook_entry_header">Picking Cells</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Yoyo, Amrinder, Patrick, Orion</li>
 +
<li>Date: May 15, 2013</li>
 +
<li>Put 50µL of Amp resistance into 50mL of Lb media</li>
 +
<li class="notebook_nested_start">Flasks:</li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li>J23100_J61002</li>
 +
<li>J23108_J61002</li>
 +
<li>J23113_J61002</li>
 +
<li>J23114_J61002</li>
 +
<li>J06702_psb1ak2</li>
 +
</ul>
 +
</li>
 +
<li>Inserted tips of E. Coli into each flask</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_fifteen">
 +
<h1 class="notebook_entry_header">Picking Cells</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Amrinder, Orion</li>
 +
<li>Date: May 16, 2013</li>
 +
<li>Put 5µL of Amp into 5mL of Lb media</li>
 +
<li class="notebook_nested_start">Flasks:</li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li>J23100_J61002</li>
 +
<li>J23108_J61002</li>
 +
<li>J23113_J61002</li>
 +
<li>J23114_J61002</li>
 +
<li>J06702_psb1ak2</li>
 +
 +
</ul>
 +
</li>
 +
<li>Inserted tips of E. Coli into each tube</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_sixteen">
 +
<h1 class="notebook_entry_header">10 Fold Miniprep</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Isaac, Patrick</li>
 +
<li>Date: May 16, 2013</li>
 +
<li>Spun down 50ml of culture in falcon tubes</li>
 +
<li>5000xg twice for 2mins to collect cells</li>
 +
<li>Resupended in 1ml solution1 2ml solution 2 and 3,5ml solution 3</li>
 +
<li>Spun down at 6000xg for 6minutes to pellet cell debries</li>
 +
<li class="notebook_nested_start">Eluted into 4 miniprep spin collumns twice to reach maximum binding effeciency<li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li>Combined eluted minipreps into one sample totalling 200µL</li>
 +
</ul>
 +
</li>
 +
 +
<li class="notebook_nested_start">Mini prep of (50µL EB):</li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li>J23100_J61002</li>
 +
<li>J23108_J61002</li>
 +
<li>J23113_J61002</li>
 +
<li>J23114_J61002</li>
 +
<li>J06702_PSB1AK2</li>
 +
</ul>
 +
</li>
 +
<li>Mini-prepped with EZ-10 columns</li>
 +
<li>Eluted 35µL</li>
 +
<li>Purified DNA in Erin’s Box</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_seventeen">
 +
<h1 class="notebook_entry_header">Agarose Gel</h1>
 +
<div class="restrict_width">
 +
<ul class="notebook_entry_list">
 +
<li>Name: Krista Fjordbotten, Yoyo Yao</li>
 +
<li>Date: May 17, 2013</li>
 +
<li class="notebook_nested_start">Restriction of:</li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li>4μL DNA</li>
 +
<li>1μL Enzyme</li>
 +
<li>2.5μL Buffer</li>
 +
<li>17.5μL Water</li>
 +
<li class="notebook_second_nested">J23100_J06702 J61002</li>
 +
<li class="notebook_second_nested">J23108_J06702 J61002</li>
 +
<li class="notebook_second_nested">J23113_J06702 J61002</li>
 +
<li class="notebook_second_nested">J23114_J06702 J61002</li>
 +
<li class="notebook_second_nested">J06702 J61002</li>
 +
</ul>
 +
</li>
 +
<li>Ladder: 2μL Ladder, 0.5μL Dye</li>
 +
<li><img src="images/conformation-gel-17-05-2013.gif" alt="Conformation Gel on May 17, 2013" /></li>
 +
<li>Conformation of all 5 of May 16, 2013 minipreps</li>
 +
</ul>
 +
</div>
 +
<ul>
 +
<li>
 +
<table class="table_three table_position">
 +
<thead>
 +
<tr>
 +
<th>Well #(from left)</th>
 +
<th>DNA Parts</th>
 +
<th>Concentration</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>2</td>
 +
<td>ladder</td>
 +
<td>2μL Ladder/0.5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>J23100_J06702 J61002</td>
 +
<td>25μL DNA/5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>J23108_J06702 J61002</td>
 +
<td>25μL DNA/5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<td>J23113_J06702 J61002</td>
 +
<td>25μL DNA/5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>J23114_J06702 J61002</td>
 +
<td>25μL DNA/5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>12</td>
 +
<td>J06702 J61002</td>
 +
<td>25μL DNA/5μL Dye</td>
 +
</tr>
 +
<tr>
 +
<td>14</td>
 +
<td>ladder</td>
 +
<td>2μL Ladder/0.5μL Dye</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_eighteen">
 +
<h1 class="notebook_entry_header">Miniprep</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Amrinder Grewal , Krista Fjordbotten</li>
 +
<li>Date: May 17, 2013</li>
 +
<li class="notebook_nested_start">Mini prep of (35µL EB):</li>
 +
<li>
 +
<ul class="notebook_nested_list">
 +
<li class="notebook_second_nested">J23100_J61002</li>
 +
<li class="notebook_second_nested">J23108_J61002</li>
 +
<li class="notebook_second_nested">J23113_J61002</li>
 +
<li class="notebook_second_nested">J23114_J61002</li>
 +
<li>Mini-prepped with EZ-10 columns</li>
 +
<li>Eluted 35µL</li>
 +
<li>Purified DNA in Erin’s Box</li>
 +
 +
</ul>
 +
</li>
 +
<li>Inserted tips of E. Coli into each flask</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_nineteen">
 +
<h1 class="notebook_entry_header">Glycerol Stock</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Name: Amrinder Grewal , Krista Fjordbotten, Yoyo Yao, Isaac Ward</li>
 +
<li>Date: May 17, 2013</li>
 +
<li class="notebook_nested_start">Glycerol Stocked:</li>
 +
<ul>
 +
<li>J23100_J61002</li>
 +
<li>J23108_J61002</li>
 +
<li>J23113_J61002</li>
 +
<li>J23114_J61002</li>
 +
</ul>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
</body>
 +
</html>

Revision as of 00:05, 7 June 2013

Ligations

  • Name: Yoyo Yao
  • Date: May 1, 2013
  • Ligation of Parts – Upstream, Ligated at sites S & P:
    • J23100
    • J23113
    • J23108 +
    • J23108 –
    • All promoters already attached to Destination Plasmid J61002
  • Downstream, Ligated at sites X & P
    • J23100

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Restriction Digest

  • Name: Mackenzie
  • Date: May 4, 2013
  • 35µL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
  • Master Mix
    Milliq 36µl
    BSA 45µl
    10x buffer 2 NEB 45µl

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1% Agarose Gel of Promoter Constructs

  • Name: Mackenzie
  • Date: May 4, 2013
  • Lane # Contents Volume
    1 1KB LADDER 6µL
    2 J23100_J61002 25µL
    3 J23100_J61002 25µL
    4 J23100_J61002 25µL
    5 J23100_J61002 25µL
    6 J23108_J61002 25µL
    7 J23108_J61002 25µL
    8 J23108_J61002 25µL
    9 J23108_J61002 25µL
    10 J23113_J61002 25µL
    11 J23113_J61002 25µL
    12 J23113_J61002 25µL
    13 J23113_J61002 25µL
    14 J23114_J61002 25µL
    15 J23114_J61002 25µL
    16 J23114_J61002 25µL
    17 J23114_J61002 25µL

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Gel Extraction

  • Date: May 4, 2013
  • J23108 J61002
  • J23113 J61002
  • J23114 J61002
  • J23100 J61002
  • Eluted 50 µL

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Transformation

  • Name: Mackenzie
  • Date: May 5, 2013
  • Tube # Part # Plasmid Volume of Competent Cells
    1 J23100+J06702 J61002 20µL
    2 J23108+J06702 J61002 20µL
    3 J23113+J06702 J61002 20µL
    4 J23108-J06702 J61002 20µL

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Restriction

  • Name: Chris Isaac
  • Date: May 7, 2013
  • Cut with ‘X’ and ‘P’
  • J06702 – RBS-mcherry-DT

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Gel Extraction

  • Name: Katie T. & Elaine B.
  • Date: May 7, 2013
  • J06702
  • Eluted at 30µL
  • Gel Extraction Done on May 7, 2013

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Gel Extracted Parts on Conformation Gel

  • 1µl of 1kb Ladder ng/0.5µg
  • 2µl DNA in all samples
  • Gel Extracted Parts on Conformation Gel Image
    Lane # Content
    1 Ladder
    2 J06702
    3 J23113–J61002 mc05/04/13mdy
    4 J23108–J61002 mc05/04/13
    5 J23100–J61002 mc05/04/13
    6 J23114–J61002 mc05/04/13
    7 Ladder
    8 J23100–J 61002 EK150413dmy
    9 J23108–J61002 EK150413
    10 J23113–J61002EK150413
    11 J23114 –J61002 EK150413

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Gel Extraction

  • Name: Carissa K.
  • Date: May 8, 2013
  • 2ul of J06702 mix with 4ul of each; J23100, J23108, J23113, J23114 all in plasmid J61002 (amp)
  • In the Thermo-cycler at 16 degrees Celsius overnight followed by heat kill.

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PCR of Ligation Mixture(PCR to verify insert in clone vector)

  • Name: Chris Isaac
  • Date: May 9, 2013
  • X7 Master Mix, Taq polymerase
  • J23100_J61002 (control)
  • J06702_pSB1AK2 (control)
  • J23100_J61002_J06702
  • J23108_J601002_J06702
  • J23113_J61002_J06702
  • J23114_J61002_J06702

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Restriction of PCR Ligations Gel Conformation

  • Name: Patrick O'
  • Date: May 13, 2013
  • Restriction of May 9th pcr ligation at E and P and ran on 1.0% Gel (refreshed ETH Bromide)
  • Ladder lane 2
  • 2space
  • J06702_pSB1AK2 (control)
  • 2spaces
  • J23100_J61002 (control)
  • spaces
  • J23100_J61002_J06702
  • J23108_J601002_J06702
  • J23113_J61002_J06702
  • J23114_J61002_J06702
  • Ladder
  • Conclusion: Ligations were not Successful
  • Restriction of PCR Ligations Gel Conformation Image

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Transformation of Ligations

  • Date: May 14, 2013
  • Transformed ligation of J06702 with various promoters in plasmid J61002 : J23100, J23108, J23113, J23114 From May 8, 2013.
  • Transformed 3ul ligation mix into 30ul of Dh5 alpha competent cells, used puc19 control. Heat shocked for 30second at 42 degrees Celsius added 200ul warm LB, incubated at 37 for 1 hour, Plated 100ul on amp LB Agar plates. Overnight at 37degrees Celsius.

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Ligation Round 2 of May 14th Gel Extracted Parts

  • 11ul dh20
  • 2ul t4 dna ligase buffer
  • 4ul DNA J06702
  • 2ul DNA J23100, J23108, J23113, J23114 in plasmid J61002
  • 1ul T4 dna Ligase
  • Into the Thermo-cycler overnight at 16 degrees Celsius.

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Picking Cells

  • Name: Yoyo, Amrinder, Patrick, Orion
  • Date: May 15, 2013
  • Put 50µL of Amp resistance into 50mL of Lb media
  • Flasks:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_psb1ak2
  • Inserted tips of E. Coli into each flask

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Picking Cells

  • Name: Amrinder, Orion
  • Date: May 16, 2013
  • Put 5µL of Amp into 5mL of Lb media
  • Flasks:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_psb1ak2
  • Inserted tips of E. Coli into each tube

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10 Fold Miniprep

  • Name: Isaac, Patrick
  • Date: May 16, 2013
  • Spun down 50ml of culture in falcon tubes
  • 5000xg twice for 2mins to collect cells
  • Resupended in 1ml solution1 2ml solution 2 and 3,5ml solution 3
  • Spun down at 6000xg for 6minutes to pellet cell debries
  • Eluted into 4 miniprep spin collumns twice to reach maximum binding effeciency
    • Combined eluted minipreps into one sample totalling 200µL
  • Mini prep of (50µL EB):
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • J06702_PSB1AK2
  • Mini-prepped with EZ-10 columns
  • Eluted 35µL
  • Purified DNA in Erin’s Box

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Agarose Gel

  • Name: Krista Fjordbotten, Yoyo Yao
  • Date: May 17, 2013
  • Restriction of:
    • 4μL DNA
    • 1μL Enzyme
    • 2.5μL Buffer
    • 17.5μL Water
    • J23100_J06702 J61002
    • J23108_J06702 J61002
    • J23113_J06702 J61002
    • J23114_J06702 J61002
    • J06702 J61002
  • Ladder: 2μL Ladder, 0.5μL Dye
  • Conformation Gel on May 17, 2013
  • Conformation of all 5 of May 16, 2013 minipreps
  • Well #(from left) DNA Parts Concentration
    2 ladder 2μL Ladder/0.5μL Dye
    4 J23100_J06702 J61002 25μL DNA/5μL Dye
    6 J23108_J06702 J61002 25μL DNA/5μL Dye
    8 J23113_J06702 J61002 25μL DNA/5μL Dye
    10 J23114_J06702 J61002 25μL DNA/5μL Dye
    12 J06702 J61002 25μL DNA/5μL Dye
    14 ladder 2μL Ladder/0.5μL Dye

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Miniprep

  • Name: Amrinder Grewal , Krista Fjordbotten
  • Date: May 17, 2013
  • Mini prep of (35µL EB):
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002
    • Mini-prepped with EZ-10 columns
    • Eluted 35µL
    • Purified DNA in Erin’s Box
  • Inserted tips of E. Coli into each flask

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Glycerol Stock

  • Name: Amrinder Grewal , Krista Fjordbotten, Yoyo Yao, Isaac Ward
  • Date: May 17, 2013
  • Glycerol Stocked:
    • J23100_J61002
    • J23108_J61002
    • J23113_J61002
    • J23114_J61002

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Retrieved from "http://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may"