Team:Lethbridge Canada/notebook june

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<ul>
<ul>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project">Description</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project">Description</a></li>
 +
                                                <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project#video_oxy">Visual Modeling</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/math">Math Model</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/math">Math Model</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/results">Results</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/wikifreeze">Wikifreeze</a></li>
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">Notebook: April</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">Notebook: April</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">Notebook: May</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">Notebook: May</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june">Notebook: June</a></li>
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/videos">Videos</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/videos">Videos</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/surveys">Parent Surveys</a></li>
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/surveys">Parent Surveys</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/novel_study">Novel Study</a></li>
</ul>
</ul>
</li>
</li>
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<div id="mainContent">
<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="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">March</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_one">March 12, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_two">March 13, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_three">March 13, 2013: Miniprep and Glycerol Stock</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_four">March 15, 2013: Restriction Digestion</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_five">March 16, 2013: Agarose Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_six">March 18, 2013: Agarose Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_seven">March 19, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_eight">March 20, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_nine">March 21, 2013: Miniprep and Glycerol Stock</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_ten">March 27, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_eleven">March 28, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_twelve">March 28, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_thirteen">March 29, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_fourteen">March 29, 2013: Picking Cells of -80 Glycerol Stock</a></li>
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</ul>
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<h1 class="heading_first">Personal Safety:</h1>
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<ul id="notebook_april_links">
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<p>When in the lab, each member of our team is careful to use gloves, goggles, and lab coats in order to protect us from any
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<li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">April</a></li>
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chemicals or specimens that might escape their containers. Although if some corrosive or other harmful chemical were to spill
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_one">April 2, 2013: Restriction - Ligation</a></li>
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on the skin or in the eye of any person in the lab, there is an eye wash station and an emergency shower located conveniently
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_two">April 2, 2013: Ligation</a></li>
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in the lab. On the first day that our team was shown the lab, the locations of each of these safety measures were made known
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_three">April 3, 2013: Transformation</a></li>
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to us. In addition to these, we were also shown the binder where the MSDS (Material Safety Data Sheet) for each chemical in  
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_four">April 4, 2013: Re-Plating Transformation</a></li>
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the lab is kept so that we would be able to handle and dispose of the chemicals properly.</p>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_five">April 5, 2013: Miniprep of ON Cultures</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_six">April 5, 2013: UV Spectroscopy DNA Concentrations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_seven">April 6, 2013: Miniprep From Ligations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_eight">April 7, 2013: PCR</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_nine">April 8, 2013: Restriction Digestion of PCR Parts</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_ten">April 8, 2013: 3% Agarose Gel of Digested PCR Parts</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_eleven">April 8, 2013: Gel Extraction</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twelve">April 9, 2013: Repeat PCR</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_thirteen">April 9, 2013: Picking Cell Colonies (Cultures)</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_fourteen">April 10, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_fifteen">April 10, 2013: Restriction</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_sixteen">April 10, 2013: Agarose Gel of PCR 1%</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_seventeen">April 11, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_eighteen">April 12, 2013: Restriction Digestion</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_nineteen">April 12, 2013: PCR and Cell Reculturization</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty">April 12, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_one">April 14, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_two">April 14, 2013: 1% Agarose Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_six">April 15, 2013: Restriction/Digestion</a></li>
 +
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_seven">April 15, 2013: Restriction/Digestion</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_three">April 15, 2013: 1% Agarose Gel of Promoters</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_twenty_four">April 17, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#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="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">May</a></li>
 +
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_one">May 1, 2013: Ligation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_two">May 4, 2013: Restriction Digest</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_three">May 4, 2013: 1% Agarose Gel of Promoter Constructs</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_four">May 4, 2013: Gel Extractions</a></li>
 +
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_five">May 5, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_six">May 7, 2013: Restriction</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_seven">May 7, 2013: Gel Extraction</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_eight">May 7, 2013: Gel Extracted on Conformation Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_nine">May 8, 2013: Ligations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_ten">May 9, 2013: PCR of Ligation Mixture</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_eleven">May 13, 2013: Restriction of PCR Ligations Gel Conformation</a></li>
 +
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twelve">May 14, 2013: Transformation of Ligations</a></li>
 +
<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirteen">May 15, 2013: Ligation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_fourteen">May 15, 2013: Picking Cells</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_fifteen">May 16, 2013: Picking Cells</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_sixteen">May 16, 2013: 10 Fold Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_seventeen">May 17, 2013: Agarose Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_eighteen">May 17, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_nineteen">May 17, 2013: Glycerol Stock</a></li>
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        <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_one">May 22, 2013: Ligation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_two">May 22, 2013: Transformations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_three">May 23, 2013: Transformations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_four">May 23, 2013: Picked Oxytocin and Transformations</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_five">May 24, 2013: Glycerol Stock</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_six">May 24, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_seven">May 26, 2013: Miniprep</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_eight">May 26, 2013: Restriction Digestion</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twenty_nine">May 26, 2013: Agarose gel of Digestion</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty">May 26, 2013: Digestion Protocol</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_one">May 31, 2013: Agarose Gel</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_two">May 31, 2013: Everything</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_thirty_three">May 31, 2013: Everything</a></li>
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</ul>
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<ul id="notebook_june_links">
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<li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june">June</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_one">June 1, 2013: Restrictions</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_two">June 2, 2013: Transformation</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_three">June 4, 2013: Picked Colonies</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_four">June 10, 2013: Sent constructs for sequencing</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_five">June 13, 2013: Transformation of OXY into BL21</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_six">June 13, 2013: Restricted and Ligated and Transformed</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_seven">June 13, 2013: Picked Cells</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_eight">June 15, 2013: Ni-Sepharose Purification</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_nine">June 18, 2013: Tris-Tricine PAGE</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_ten">June 21, 2013: Anti-His Slot Blot</a></li>
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</ul>
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</div>
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</div>
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<div class="pull_banner_left">
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<img src="https://static.igem.org/mediawiki/2013hs/2/21/Lethbridge_hs_igem_2013_banner_notebookjune.png">
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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">Restrictions</h1>
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<ul class="notebook_entry_list">
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<li>Restricted (total 60µL DNA) K314100 in pSB1C3 with Spe1 and Pst1</li>
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<li>Restricted (total 60µL DNA) Oxytocin in puc19a with Xba1 and Pst1</li>
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<li>Restricted (total 60µL DNA) NEC1puc19a with Xba1 and Pst1</li>
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<li>Set 12 µL aside from each reaction and gel extracted (GEX) the remaining  48µL</li>
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<li>Combined heat killed restriction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80°C </li>
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<li>K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)</li>
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<li>K314100 – pSB1C3 (s,p), with: NEC1 (x,p)</li>
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<li>GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)</li>
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<li>GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)</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">Transformation</h1>
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<ul class="notebook_entry_list">
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<li class="notebook_nested_start">Transformed ligations into 20µL high efficiency DH5α cells and 50µL lab made competent DH5α</li>
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<li><ul class="notebook_nested_list">
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<li>K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)</li>
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<li>K314100 – pSB1C3 (s,p), with: NEC1 (x,p)</li>
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<li>GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)</li>
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<li>GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)</li>
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</ul>
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</li>
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<li>Plated on CAM plates</li>
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<li>Results: Only the 20µL non gel extracted DH5α E. coli high efficiency cells grew</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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<h1 class="notebook_entry_header">Picked Colonies</h1>
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<ul class="notebook_entry_list">
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<li>Date: June 4, 2013</li>
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<li>Picked 10 colonies from each plate</li>
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<li>Grew over night in LB and mini prepped</li>
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<li>PCR-ed with VF2 and VR primers, no results as shown</li>
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<li><img src="https://static.igem.org/mediawiki/2013hs/b/bd/Leth_hs_igem_june_10_1_colonies.png" alt="Gel on June 4, 2013"></li>
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<li>  Picked 6 Oxytocin colonies and 5 NEC1 colonies grew overnight in LB – mini prepped and restricted all colonies picked so far at E, P cute sites and ran on gel, results concluded as ligations did not work since a band is seen at ~500 bp size of the promoter construct K314100 but not with oxytocin or NEC1</li>
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<li><img src="https://static.igem.org/mediawiki/2013hs/7/77/Leth_hs_igem_june_10_2_colonies.png" alt="Gel on June 4, 2013"></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">Picked Colonies</h1>
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<ul class="notebook_entry_list">
 +
<li>Date: June 10, 2013</li>
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<li class="notebook_nested_start">Sent 10µL DNA with 5µL of 5µM VF2 primer to genewize</li>
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<li><ul class="notebook_nested_list">
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<li>J23100-J06702</li>
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<li>J23108-J06702</li>
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<li>J23113-J06702</li>
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<li>K314100-oxytocin neurophysin I </li>
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</ul>
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</li>
 +
<li class="notebook_nested_start">June 12th results arrived</li>
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<li><ul class="notebook_nested_list">
 +
<li>J23100-J06702- positive for J06702 but no promoter found</li>
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<li>J23108-J06702- positive for J06702 but no promoter found</li>
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<li>J23113-J06702- positive for J06702 but no promoter found</li>
 +
<li>K314100-oxytocin neurophysin I – Positive results full sequence found</li>
 +
</ul>
 +
</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_five">
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<h1 class="notebook_entry_header">Transformation of OXY into BL21</h1>
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<ul class="notebook_entry_list">
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<li>Date: June 13, 2013</li>
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<li>Transform mini prepped plasmid DNA of K314100–Oxytocin Neurophysin I  in pSB1C3 into 15µL of Bl21 E.coli cells </li>
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<li>Let grow over night at 37°C on CAM plates</li>
 +
</ul>
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 +
</div>
 +
</div>
 +
 
 +
<div class="notebook_entry">
 +
<div id="entry_six">
 +
<h1 class="notebook_entry_header">Restricted and Ligated and Transformed</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: June 13, 2013</li>
 +
<li>Restricted K314100 Spe1 and Pst1 and NEC1 at Xba1 and Pst1 for 1 hour at 37°C followed by 20 min heat kill at 80°C</li>
 +
<li>Mixed restrictions in ratio of 10µL NEC1 and 2µL k314100 in pSB1C3  let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80°C</li>
 +
<li>Transformed 2µL into 20µL of DH5α E.coli highly competent cells, incubated overnight at 37°C</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">Picked Cells</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: June 13, 2013</li>
 +
<li>K314100–Oxytocin neurophysin 1 in pSB1C3 grew in Bl21 from last nights transformation, picked 4 colonies and grew up in 5ml of LB overnight at 37°C with shaking</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 
 +
<div class="notebook_entry">
 +
<div id="entry_eight">
 +
<h1 class="notebook_entry_header">Ni-Sepharose Purification</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: June 15, 2013</li>
 +
<li>Grew 250mL culture of K314100 – Oxytocin neurophysin in psb1c3 overnight at 37&deg;C with shaking (June 14)</li>
 +
<li>Purified preprooxyphysin with Ni-Sapharose beads according to Wieden Protocol</li>
 +
<li>Eluted overnight</li>
 +
<li>Will confirm with tris-tricine PAGE</li>
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 +
<div class="notebook_entry">
 +
<div id="entry_nine">
 +
<h1 class="notebook_entry_header">Tris-Tricine PAGE</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: June 18, 2013</li>
 +
<li>Prepro-oxyphysin can be seen at the bottom of lanes 5 and 7</li>
 +
<li>
 +
<table>
 +
<tr>
 +
<th>Lane</th>
 +
<th>Contents</th>
 +
<th>Volume</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>Protein Molecular Weight Marker</td>
 +
<td>20&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>Cbf5-Nop10-Gar1</td>
 +
<td>20&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>Buffer A wash</td>
 +
<td>30&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>Buffer B wash</td>
 +
<td>30&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>Buffer E Wash</td>
 +
<td>30&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>S-30 s/N</td>
 +
<td>30&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<td>Buffer E – Concentrated Preprooxyphysin</td>
 +
<td>30&mu;L</td>
 +
</tr>
 +
</table>
 +
</li>
 +
</ul>
 +
<img class="june_et_img" src="https://static.igem.org/mediawiki/2013hs/3/34/Leth_hs_2013_june_18_sheet_1.png" alt=" " />
 +
<img class="june_et_img" src="https://static.igem.org/mediawiki/2013hs/b/bc/Leth_hs_2013_june_18_sheet_2.png" alt=" " />
 +
<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">Anti-His Slot Blot</h1>
 +
<ul class="notebook_entry_list">
 +
<li>Date: June 21, 2013</li>
 +
<div style="display:block">
 +
<li><table style="float:left;" width:242px; height:120px;>
 +
<tbody><tr>
 +
<th>Lane #</th>
 +
<th>Contents</th>
 +
</tr><tr>
-
<p>Before any member of our team was able to participate in any lab work he/she was required take and pass a WHMIS (Workplace
+
</tr><tr>
-
Hazardous Material Information System) test. Also, before beginning our project, as a team we had to observe and perform
+
<td>Lane 1:</td>
-
tutorials of the experiments that we were going to perform. Not only was this a fantastic learning experience, but also it
+
<td>100μL Oxytocin in 200μL TBS</td>
-
helped us to become more comfortable in this lab setting and therefore less likely to make a mistake and compromise our safety.
+
</tr>
-
In addition to all of this, if any person had missed the instruction on how to use a piece of equipment properly or was
+
-
unclear on a protocol, there is always an adviser in the lab with us and could explain.</p>
+
-
<h1>Environmental/Public Safety:</h1>
 
-
<p>Although it is unlikely that our specimens would unintentionally leave our lab area or that it would cause any extreme
 
-
harmful effects if it did get outside of the lab, we still took precautions. The E-coli that we work with is a non-pathogenic
 
-
strain (DH5 alpha) and is engineered not to be able to survive outside of our laboratory. Although this is also the case,
 
-
each member of our team is extremely careful to keep the specimens in a sealed container and to transport it very carefully.</p>
 
-
<p>Although there is no biosafety group, committee or review board at our institution, we were required to follow university
+
<tr>
-
risk and safety guidelines. We also deferred to the Public Health Agency of Canada – Laboratory Biosafety and Biosecurity.</p>
+
<td>Lane 2:</td>
 +
<td>200μL Oxytocin in 100μL TBS</td>
 +
</tr>
 +
 
 +
 +
<tr>
 +
<td>Lane 3:</td>
 +
<td>250μL Oxytocin in 50μL TBS</td>
 +
</tr>
 +
 
 +
 +
<tr>
 +
<td>Lane 4:</td>
 +
<td>300μL Oxytocin</td>
 +
</tr>
 +
 
 +
 +
<tr>
 +
<td>Lane 5:</td>
 +
<td>25pM TruB-His positive control</td>
 +
</tr>
 +
</tbody></table>
 +
</li>
 +
<img class="june_et_img" src="https://static.igem.org/mediawiki/2013hs/a/a1/Leth_hs_2013_oxt_histag.png" alt=" "
 +
style="float:left; width:830px; height:135px; padding-left:10px;" />
 +
</div>
 +
 
 +
<p>We used a 25pM sample of the protein TruB with a histidine tag which is seen in lane 5. In lanes 1,2,3 and 4, faint bands are seen on the slot blot. This is indicative of the histidine tag attached to Oxytocin-NeurophysinI.</p>
 +
 
 +
</ul>
 +
<p class="top_notebook"><a href="#wrapper">Back to Top</a></p>
 +
</div>
 +
</div>
 +
 
 +
</div>
</div>
</div>

Latest revision as of 03:56, 22 June 2013

Restrictions

  • Restricted (total 60µL DNA) K314100 in pSB1C3 with Spe1 and Pst1
  • Restricted (total 60µL DNA) Oxytocin in puc19a with Xba1 and Pst1
  • Restricted (total 60µL DNA) NEC1puc19a with Xba1 and Pst1
  • Set 12 µL aside from each reaction and gel extracted (GEX) the remaining 48µL
  • Combined heat killed restriction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80°C
  • K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
  • K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
  • GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
  • GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)

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Transformation

  • Transformed ligations into 20µL high efficiency DH5α cells and 50µL lab made competent DH5α
    • K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
    • K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
    • GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
    • GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)
  • Plated on CAM plates
  • Results: Only the 20µL non gel extracted DH5α E. coli high efficiency cells grew

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Picked Colonies

  • Date: June 4, 2013
  • Picked 10 colonies from each plate
  • Grew over night in LB and mini prepped
  • PCR-ed with VF2 and VR primers, no results as shown
  • Gel on June 4, 2013
  • Picked 6 Oxytocin colonies and 5 NEC1 colonies grew overnight in LB – mini prepped and restricted all colonies picked so far at E, P cute sites and ran on gel, results concluded as ligations did not work since a band is seen at ~500 bp size of the promoter construct K314100 but not with oxytocin or NEC1
  • Gel on June 4, 2013

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Picked Colonies

  • Date: June 10, 2013
  • Sent 10µL DNA with 5µL of 5µM VF2 primer to genewize
    • J23100-J06702
    • J23108-J06702
    • J23113-J06702
    • K314100-oxytocin neurophysin I
  • June 12th results arrived
    • J23100-J06702- positive for J06702 but no promoter found
    • J23108-J06702- positive for J06702 but no promoter found
    • J23113-J06702- positive for J06702 but no promoter found
    • K314100-oxytocin neurophysin I – Positive results full sequence found

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Transformation of OXY into BL21

  • Date: June 13, 2013
  • Transform mini prepped plasmid DNA of K314100–Oxytocin Neurophysin I in pSB1C3 into 15µL of Bl21 E.coli cells
  • Let grow over night at 37°C on CAM plates

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Restricted and Ligated and Transformed

  • Date: June 13, 2013
  • Restricted K314100 Spe1 and Pst1 and NEC1 at Xba1 and Pst1 for 1 hour at 37°C followed by 20 min heat kill at 80°C
  • Mixed restrictions in ratio of 10µL NEC1 and 2µL k314100 in pSB1C3 let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80°C
  • Transformed 2µL into 20µL of DH5α E.coli highly competent cells, incubated overnight at 37°C

Back to Top

Picked Cells

  • Date: June 13, 2013
  • K314100–Oxytocin neurophysin 1 in pSB1C3 grew in Bl21 from last nights transformation, picked 4 colonies and grew up in 5ml of LB overnight at 37°C with shaking

Back to Top

Ni-Sepharose Purification

  • Date: June 15, 2013
  • Grew 250mL culture of K314100 – Oxytocin neurophysin in psb1c3 overnight at 37°C with shaking (June 14)
  • Purified preprooxyphysin with Ni-Sapharose beads according to Wieden Protocol
  • Eluted overnight
  • Will confirm with tris-tricine PAGE

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Tris-Tricine PAGE

  • Date: June 18, 2013
  • Prepro-oxyphysin can be seen at the bottom of lanes 5 and 7
  • Lane Contents Volume
    1 Protein Molecular Weight Marker 20μL
    2 Cbf5-Nop10-Gar1 20μL
    3 Buffer A wash 30μL
    4 Buffer B wash 30μL
    5 Buffer E Wash 30μL
    6 S-30 s/N 30μL
    7 Buffer E – Concentrated Preprooxyphysin 30μL

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Anti-His Slot Blot

  • Date: June 21, 2013
  • Lane # Contents
    Lane 1: 100μL Oxytocin in 200μL TBS
    Lane 2: 200μL Oxytocin in 100μL TBS
    Lane 3: 250μL Oxytocin in 50μL TBS
    Lane 4: 300μL Oxytocin
    Lane 5: 25pM TruB-His positive control

    • We used a 25pM sample of the protein TruB with a histidine tag which is seen in lane 5. In lanes 1,2,3 and 4, faint bands are seen on the slot blot. This is indicative of the histidine tag attached to Oxytocin-NeurophysinI.

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