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| - | <div id="protocol">
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| - | <a href="https://2013hs.igem.org/Team:Lethbridge_Canada">
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| - | <div id="logo_all">
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| - | <img src="https://static.igem.org/mediawiki/2013hs/3/32/Leth_hs_2013_logo.png" alt="logo" />
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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="cont">
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| - | <li class="navigation_project"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project" class="strict">Project</a>
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| - | <ul>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/project">Description</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/math">Math Model</a></li>
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| - | </ul>
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| - | <li class="navigation_parts decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/parts" class="strict">Parts</a></li>
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| - | <li class="navigation_notebook decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/protocols" class="strict">Notebook</a>
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| - | <ul>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/protocols#protocol_header">Protocols</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march">Notebook: March</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april">Notebook: April</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may">Notebook: May</a></li>
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| - | </ul>
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| - | <li class="navigation_safety decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/safety" class="strict">Safety</a></li>
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| - | <li class="navigation_outreach decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/outreach" class="strict">Outreach</a>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/outreach">Overview</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/presentations">Presentations</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/interviews">Interviews</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/videos">Videos</a></li>
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| - | </ul>
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| - | <li class="navigation_team decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team" class="strict">Team</a>
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| - | <ul>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team#the_team">The Team</a></li>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/team#the_advisors">The Advisors</a></li>
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| - | </ul>
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| - | </li>
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| - | <li class="navigation_sponsors decrease_opacity"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/sponsors" class="strict">Sponsors</a></li>
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| - | </ul>
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| - | </div>
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| - | </div>
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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="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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| - |
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| - | <ul id="notebook_april_links">
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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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| - | <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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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_april#entry_two">April 2, 2013: Ligation</a></li>
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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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| - | <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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| - | <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>
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| - | <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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| - |
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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>
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| - | <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>
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| - | <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>
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| - | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_may#entry_twelve">May 14, 2013: Transformation of Ligations</a></li>
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| - | <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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| - | </ul>
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| - | </div>
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| - | </div>
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| - |
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| - |
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| - | <h1 id="protocol_header">Protocols:</h1>
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| - | <div id="protocols_content">
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| - | <div class="protocol_entry">
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| - | <div id="agarose">
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| - | <h1>Agarose Gel Electrophoresis</h1>
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| - |
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| - | <h2>Prepare the Agarose Gel:</h2>
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| - |
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| - | <ol>
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| - | <li>Weigh appropriate amount of agarose into a small Erlenmeyer flask (0.3g for 30mL small 1% gel).</li>
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| - | <li>Add desired volume of 1x TAE buffer (or 1x TBE in special cases) (30mL for small gel chamber).</li>
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| - | <li>Weight the flask and write down its total weight.</li>
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| - | <li>Boil it to resolve the agarose:
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| - | <ul>
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| - | <li>either by microwaving (without magnetic stir bar)</li>
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| - | <li>or on hot plate (with magnetic stir bar).</li>
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| - | </ul></li>
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| - | <li>Let it cool down while stirring to about 60°C (hand-warm).</li>
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| - | <li>Weight again and replace lost water.</li>
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| - | <li>Seal ends of gel plate with black wedges.</li>
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| - | <li>Cast gel into gel plate and put in the comb, wait until solid.</li>
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| - | </ol>
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| - |
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| - | <h2>Prepare Samples:</h2>
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| - |
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| - | <ol>
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| - | <li>Transfer about 0.3μg DNA into a microcentrifuge tube.</li>
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| - | <li>Add 1x TAE to 5μL.</li>
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| - | <li>Add 1μL of 6x DNA sample buffer.</li>
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| - | </ol>
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| - |
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| - | <h2>Run Electrophoresis:</h2>
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| - |
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| - | <ol>
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| - | <li>Place gel into the big chamber in correct orientation (DNA migrates to the positive pole)</li>
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| - | <li>Fill chamber with 1x TAE buffer (or 1x TBE), about 0.5cm above gel.</li>
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| - | <li>Carefully remove comb from solid gel.</li>
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| - | <li>Load the 6 μL samples into the gel slots, and load 4 μL of DNA ladder (typically 1kb ladder) in at least one gel slot.</li>
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| - | <li>Close chamber with lid in the correct orientation (black/red).</li>
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| - | <li>Connect cables to power supply.</li>
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| - | <li>Run the gel with 1–5V/cm of gel length, check for dye fronts should be at about 1/3 and 2/3 of gels (about 1h) – common lab procedure: 100V for small gel.</li>
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| - | </ol>
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| - |
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| - | <h2>Staining and Photo:</h2>
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| - |
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| - | <ol>
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| - | <li>Stain the gels for 10–20 minutes in ethidiumbromide while shaking. Attention: always wear gloves with ethidiumbromide, try not to touch the solution or the stained gel but use a kitchen spatula to handle the gel.</li>
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| - | <li>Destain the gel for a short while in water (e.g. while transporting it to take the photo).</li>
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| - | <li>Observe bands on UV illuminator and take a digital photo. Carfeful: strong UV light, wear glasses and lab coat to avoid “sun burn."</li>
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| - | <li>Clean UV illuminator.</li>
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| - | <li>Discard gel in ethidiumbromide waste.</li>
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| - | </ol>
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| - |
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| - | <h2>Buffers:</h2>
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| - |
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| - | <div id="agarose_buffers">
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| - |
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| - | <div class="border_right">
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| - | <h3>50x TAE - Dilution to 4L of 1x TAE Buffer:</h3>
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| - | <ul>
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| - | <li>242g Tris </li>
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| - | <li>80ml 50x TAE buffer</li>
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| - | <li>57.1mL acetic acid </li>
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| - | <li>fill up to 4L with MilliQ H<sub>2</sub>O</li>
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| - | <li>100mL 0.5 M EDTA pH 8.0</li>
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| - | <li>H<sub>2</sub>O to final volume of 1L</li>
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| - | </ul>
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| - | </div>
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| - |
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| - | <div class="border_right">
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| - | <h3>55x TBE:</h3>
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| - | <ul>
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| - | <li>54g Tris base - end concentration 90mM</li>
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| - | <li>27.5mL boric acid - end concentration 90mM</li>
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| - | <li>20 ml 0.5 M EDTA ph 8.0 - end concentration 1mM</li>
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| - | <li>H<sub>2</sub>O to final volume of 1L
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| - | <ul>
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| - | <li>Should be pH 8.3 without adjustment</li>
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| - | <li>Filter to delay precipitation</li>
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| - | </ul>
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| - | </li>
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| - | </ul>
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| - | </div>
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| - |
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| - | <div>
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| - | <h3>6x DNA Loading Buffer:</h3>
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| - | <ul>
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| - | <li>0.25% bromphenol blue</li>
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| - | <li>0.25% Xylencyanol</li>
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| - | <li>60% Glycerol</li>
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| - | <li>1% SDS</li>
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| - | <li>20mM EDTA</li>
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| - | <li>In H<sub>2</sub>O</li>
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| - | </ul>
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| - | </div>
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| - |
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| - | </div>
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| - |
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| - | <h2>Gene Ruler 1kb DNA ladder (Fermentas)</h2>
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| - |
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| - | <table class="protocols_table">
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| - | <tbody>
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| - | <tr>
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| - | <td rowspan="4">1:6 Dilution</td>
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| - | </tr>
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| - | <tr><td>100μLDNA ladder (0.5 μg/μL)</td></tr>
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| - | <tr><td>100μL6x Loading Dye</td></tr>
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| - | <tr><td>400μMilliQ H<sub>2</sub>O</td></tr>
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| - | </tbody>
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| - | </table>
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| - |
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| - | <p>Results in 0.08333μg/μL, i.e. 0.5μg in 6μL as recommended by Fermentas</p>
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| - |
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| - | <h2>Gel Concentration to Separate Various Sizes of DNA:</h2>
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| - |
| |
| - | <table class="protocols_table">
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| - | <thead>
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| - | <tr>
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| - | <th>Agarose in % (w/v)</th>
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| - | <th>Optimal for Linear Double-Stranded DNA (size in kb)</th>
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| - | </tr>
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| - | </thead>
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| - | <tbody>
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| - | <tr>
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| - | <td>0.3</td>
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| - | <td>5 - 60</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>0.6</td>
| |
| - | <td>1 - 20</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>0.7</td>
| |
| - | <td>0.8 - 10</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>0.9</td>
| |
| - | <td>0.5 - 7</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>1.2</td>
| |
| - | <td>0.4 - 6</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>1.5</td>
| |
| - | <td>0.2 - 3</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>2.0</td>
| |
| - | <td>0.1 - 2</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>3.0*</td>
| |
| - | <td>optimal for 50–100bp fragments, e.g. templates for in vitro transcription</td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - |
| |
| - | <p class="italic">* For gels of more than 2%, use 3 parts “cheap” agarose and 1 part Top Vision Agarose, e.g. 0.9g cheap agarose + 0.3g Top Vision (not low melting) agarose for 40mL of 3.% gel</p>
| |
| - | </div>
| |
| - | </div>
| |
| - |
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Agarose Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Overnight Cultures Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - |
| |
| - | <div class="protocol_entry">
| |
| - | <div id="overnight_cultures">
| |
| - |
| |
| - | <h1>Overnight Cultures</h1>
| |
| - |
| |
| - | <h2>Overnight Culture of Bacteria for Plasmid Purification:</h2>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Using proper Aseptic technique, sterilely transfer 5mL of LB medium into a sterile culture tube or falcon tube.</li>
| |
| - | <li>Thaw the appropriate antibiotic and pipette in the amount needed.</li>
| |
| - | <li>Sterilize the inoculating loop using flame and allow cooling then carefully picking one colony from a plate or scraping some media from a glycerol stock and transferring the cells to the media by swirling the inoculating loop.</li>
| |
| - | <li>Secure the lids of the vessel and incubate overnight with shaking at appropriate temp (usually 37°C)</li>
| |
| - | </ol>
| |
| - |
| |
| - | <h2>Plasmid Purification:</h2>
| |
| - |
| |
| - | <ol>
| |
| - | <li>If a glycerol stock of the bacteria has not previously been made do so now by cutting the end off of a 1000μL tip and pipette 200μL of 100% autoclaved glycerol into a cryomicrofuge tube.
| |
| - | <ul>
| |
| - | <li>Glycerol is very viscus and this will take practice to transfer 200μL accurately.</li>
| |
| - | <li>Do not pipet from the common glycerol bottle! Always pour some glycerol into a microfuge tube and keep your stock separate</li>
| |
| - | </ul>
| |
| - | </li>
| |
| - | <li>Pipette 800μLo f overnight culture into the cryovial and mix by swirling the tip and carefully pipetting up and down. Be careful not to suck any media into the barrel of you pipette.</li>
| |
| - | <li>Freeze the cells in liquid nitrogen and store in the -800°C freezer</li>
| |
| - | <li>Transfer the remaining bacteria into a falcon tube and centrifuge at 5000XG for 7 minutes. Alternately you can pipette cells 1.5 mL at a time in a microfuge tube at max speed for 1.5 minutes.</li>
| |
| - | <li>Remove the supernatant and dispose of in the bacteria waste vessel and proceed to the miniprep protocol found in the miniprep kits.</li>
| |
| - | </ol>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Overnight Cultures Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Restriction Digestion Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - |
| |
| - | <div class="protocol_entry">
| |
| - | <div id="restriction_digestion">
| |
| - |
| |
| - | <h1>Restriction Digestion</h1>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Pipet in this order into a 1.5ml microcentrifuge tube:
| |
| - | <ol>
| |
| - | <li>MilliQ H<sub>2</sub>O - final total volume: 20μL</li>
| |
| - | <li>10x Buffer (corresponding to enzyme) - 2μL</li>
| |
| - | <li>Plasmid DNA (volume according to concentration) 2μg(end concentration < 0.3μg/μL)</li>
| |
| - | <li>Restriction enzyme - 1-2U/μg DNA
| |
| - | <ul>
| |
| - | <li>(Volume according to concentration stated on enzyme tube)</li>
| |
| - | <li>(Don’t pipet less than 0.25μL)</li>
| |
| - | </ul>
| |
| - | </li>
| |
| - | <li>Attention with handling restriction enzymes: take them only immediately before use out of -20°C fridge, store on ice, put back into fridge as soon as possible.</li>
| |
| - | </ol>
| |
| - | </li>
| |
| - | <li>Incubate at optimal for 1 hour (some enzymes need longer times E.g. SalI) (incubation at 37°C in water bath, but e.g. for SmaI 25-30°C).</li>
| |
| - | <li>Take sample for agarose gel or store for short-term on ice or for long-term at -20°C.</li>
| |
| - | </ol>
| |
| - |
| |
| - | <h2>Preparative:</h2>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Parts:
| |
| - | <ul>
| |
| - | <li>MilliQ - 30μL</li>
| |
| - | <li>10x Buffer (corresponding to enzyme) - 1/10 of final volume 3μL</li>
| |
| - | <li>Plasmid DNA - 0.2μg/μL, 6μg</li>
| |
| - | <li>Restriction enzyme - 1U/μg DNA</li>
| |
| - | </ul>
| |
| - | </li>
| |
| - | <li>Incubate 2 hours at 37°C</li>
| |
| - | <li>Note:
| |
| - | <ul>
| |
| - | <li>SmaI needs to be incubated at 25-30°C (only 50% activity at 37°C)</li>
| |
| - | <li>SalI needs long incubation times (>4h)</li>
| |
| - | </ul>
| |
| - | </li>
| |
| - | </ol>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Restriction Digestion Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Miniprep Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - |
| |
| - |
| |
| - | <div class="protocol_entry">
| |
| - | <div id="miniprep">
| |
| - |
| |
| - | <h1>Miniprep Using EZ-10 Spin Column Kits (High Copy Number Plasmid)</h1>
| |
| - |
| |
| - | <h2>Procedure:</h2>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Warm Elution Buffer in incubator or in water bath at 37-50°C.</li>
| |
| - | <li>Add 1.5mL of the overnight culture to a 1.5mL microcentrifuge tube and centrifuge at 12000rpm for 2 minutes. Drain supernatant and repeat until all of the overnight culture is used.</li>
| |
| - | <li>Add 100μL of Solution 1 to the cell pellet, mix by pipetting, let stand 1 minute.</li>
| |
| - | <li>Add 200μL of Solution 2 to the mixture, mix gently by inverting the tube 4-6 times and let stand for 1 minute. Do not vortex.</li>
| |
| - | <li>Add 350μL of Solution 3 and mix gently by inverting the tube 4-6 times and let stand for 1 minute.</li>
| |
| - | <li>Centrifuge at 12000rpm for 5 minutes.</li>
| |
| - | <li>Transfer supernatant to an EZ-10 Spin Column and centrifuge at 10000 rpm for 2 minutes.</li>
| |
| - | <li>Discard the flow through in the tube. Add 500μL of Wash Solution to the column and centrifuge at 10000 rpm for 2 minutes.</li>
| |
| - | <li>Repeat wash procedure in Step 7.</li>
| |
| - | <li>Discard the flow though in the collection tube. Centrifuge at 10000rpm for an additional minute to remove any residual wash solution.</li>
| |
| - | <li>Transfer the column to a clean, labelled 1.5 mL microcentrifuge tube. Add 50μL of Elution Buffer to the centre part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10000 rpm for 2 minutes.</li>
| |
| - | <li>Store purified DNA at -20°C.</li>
| |
| - | </ol>
| |
| - |
| |
| - | <h2>Labeling:</h2>
| |
| - |
| |
| - | <p>All tubes are to be labeled with this information:</p>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Name</li>
| |
| - | <li>Date</li>
| |
| - | <li>Antibiotic</li>
| |
| - | <li>Species</li>
| |
| - | <li>Strain</li>
| |
| - | <li>Part Number and Plasmid</li>
| |
| - | </ul>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Miniprep Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Transformation Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <div class="protocol_entry">
| |
| - | <div id="transformation">
| |
| - |
| |
| - | <h1>Transformation of Competent Cells</h1>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Thaw 20 μL of pre aliquotted cells (Dh5α or BL21 DE3) on ice. Competent cells are stored at -80°C. (Often cells are frozen as 50 μL aliquots – split under sterile conditions for 2 transformations.)</li>
| |
| - | <li>Gently pipet maximum 2.0μL (better 1.8μL) of DNA into the competent cells and pipet once up and down to rinse the tip.
| |
| - | <ul>
| |
| - | <li>Never use more DNA than 10% of the volume of the competent cells, otherwise the cells get destroyed by osmotic shock.</li>
| |
| - | </ul>
| |
| - | </li>
| |
| - | <li>Mix the DNA into the cells by swirling the tip in the solution.</li>
| |
| - | <li>Incubate the cells on ice for 30 minutes.</li>
| |
| - | <li>Heat shock the cells in a water bath at 42°C for exactly 45 seconds.</li>
| |
| - | <li>Incubate the cells on ice 1 minute.</li>
| |
| - | <li>Add 250 μL of sterile media to the cells and incubate at 37°C for 1 hour with shaking (tape microcentrifuge tube in shaking incubator).</li>
| |
| - | <li>Label the LB plates on the outside perimeter:
| |
| - | <ol id="trans_labeling">
| |
| - | <li>Your name</li>
| |
| - | <li>Date</li>
| |
| - | <li>Cell strain (e.g. DH5α, BL21DE3 etc.)</li>
| |
| - | <li>Plasmid</li>
| |
| - | <li>Volume plated</li>
| |
| - | </ol>
| |
| - | </li>
| |
| - | <li>Plate 100 μL and 50 μL on pre-warmed LB plates containing the appropriate antibiotic. For ligations and mutagenesis: plate all 250 μL on (1 or) 2 plate.</li>
| |
| - | <li>Leave plate for 10-15 minutes to soak the cell suspension into the agar.</li>
| |
| - | <li>Flip plate over (agar on top).</li>
| |
| - | <li>Incubate the plates in the 37°C oven overnight.</li>
| |
| - | <li>Keep the remaining solution in the 4°C fridge overnight until transformation has been confirmed.</li>
| |
| - | </ol>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Transformation Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Osmotic Shock Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <div class="protocol_entry">
| |
| - | <div id="osmotic_shock">
| |
| - |
| |
| - | <h1>Osmotic Shock Procedure:</h1>
| |
| - |
| |
| - | <h2>Osmotic Shock Procedure:</h2>
| |
| - |
| |
| - | <p><i>Note: Osmotic shock procedure should be performed on cells immediately after harvesting (i.e. do not freeze cells).</i></p>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Wash harvested cells with 40 mL ice-cold Wash Buffer (30 mM NaCl, 10 mM Tris-HCl (pH 7.5 @ 4°C)) per gram of harvested cells. After washing, pellet cells by centrifugation (5000 × g, 4°C, 10 min) and remove supernatant.</li>
| |
| - | <li>Repeat step 1) once.</li>
| |
| - | <li>Resuspend cell pellet in 40 mL Buffer A (33 mM Tris HCl (pH 7.5 @ 20°C)) per gram of harvested cells (perform at room temperature).</li>
| |
| - | <li>Rapidly mix suspension with 40 mL Stage I Buffer (40% sucrose, 33 mM Tris-HCl (pH 7.5 @ 20°C), 100 μM ethylenediaminetetraacetic acid (EDTA)) per gram of harvested cells (perform at room temperature).</li>
| |
| - | <li>Incubate mixture on a rotary shaker (180 RPM) at room temperature for 10 minutes.</li>
| |
| - | <li>Centrifuge mixture (13 000 × g, 4°C, 10 min) and remove supernatant.</li>
| |
| - | <li>Rapidly resuspend cell pellet in 80 mL ice-cold Stage II Buffer (500 μM MgCl<sub>2</sub>) per gram of harvested cells.</li>
| |
| - | <li>Incubate mixture on a rotary shaker (180 RPM) at 4°C for 10 minutes. </li>
| |
| - | <li>Centrifuge samples (13 000 × g, 4°C, 10 min) and remove supernatant containing periplasmic proteins.</li>
| |
| - | </ol>
| |
| - |
| |
| - | <h2>Characterization:</h2>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Characterize samples collected throughout the purification by SDS-PAGE. There are also protein markers present in E. coli that can be used to determine the success of the Osmotic Shock Procedure. These markers are β-galactosidase (Cytosolic: 116 kDa) and β-lactamase (Periplasmic: 29 kDa).</li>
| |
| - | <li>After Step 9) of the procedure is completed, a wet mount the final cell pellet can be analyzed by light microscopy. The majority of the cells should be intact (i.e. there should be a minimal amount of lysed ghost cells)</li>
| |
| - | </ul>
| |
| - |
| |
| - | <h2>References:</h2>
| |
| - |
| |
| - | <ul>
| |
| - | <li>Gray, G. L., Baldridge, J. S., McKeown, K. S., Heyneker, H. L., & Chang, C. N. (1985). Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable. Gene, 39(2), 247-254</li>
| |
| - | <li>Nossal, N. G., & Heppel, L. A. (1966). The release of enzymes by osmotic shock from Escherichia coli in exponential phase. Journal of Biological Chemistry, 241(13), 3055-3062</li>
| |
| - | <li>Willsky, G. R., & Malamy, M. H. (1976). Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli. Journal of Bacteriology, 127(1), 595-609</li>
| |
| - | </ul>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| End of Osmotic Shock Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <!-- |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| Beginning of Tris-tricine Gel Section |-∙-∙-∙-∙-∙-∙-∙-∙-∙-∙-| -->
| |
| - | <div class="protocol_entry">
| |
| - | <div id="tris_tricine">
| |
| - |
| |
| - | <h1>Tris-tricine Gel:</h1>
| |
| - |
| |
| - | <p>These gels use tricine instead of glycine that is used in SDS-PAGE. Tricine improves separation and resolution of small proteins. Note, that instead of using one running buffer, there are two different buffers for cathode and anode. All other steps are the same as in the general SDS-PAGE protocol.</p>
| |
| - |
| |
| - | <h2>Buffers:</h2>
| |
| - |
| |
| - | <h3>3X Gel Buffer:</h3>
| |
| - | <ul>
| |
| - | <li>3M Tris-HCl (pH 8.45)</li>
| |
| - | <li>0.3% SDS</li>
| |
| - | </ul>
| |
| - |
| |
| - | <h3>10X Cathode Buffer:</h3>
| |
| - | <ul>
| |
| - | <li>1M Tris (pH 8.25) *No need to correct the pH</li>
| |
| - | <li>1M tricine </li>
| |
| - | <li>1% SDS</li>
| |
| - | </ul>
| |
| - |
| |
| - | <h3>10X Anode Buffer:</h3>
| |
| - | <ul>
| |
| - | <li>2M Tris-HCl (pH 8.9)</li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>(You need ~650 mL 1X anode buffer and ~180 mL 1X cathode buffer for each run)</p>
| |
| - |
| |
| - | <h2>Gel Composition:</h2>
| |
| - |
| |
| - | <table class="table_3">
| |
| - | <tr>
| |
| - | <th> </th>
| |
| - | <th>Resolving Gel (16.5%)</th>
| |
| - | <th>Stacking Gel (4%)</th>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Acrylamide (29:1) (40%)</td>
| |
| - | <td>2mL</td>
| |
| - | <td>0.3mL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>3X Gel Buffer</td>
| |
| - | <td>1.670mL</td>
| |
| - | <td>0.75mL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>Glycerol (50%)</td>
| |
| - | <td>1.334mL</td>
| |
| - | <td>0.00mL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>MilliQ Water</td>
| |
| - | <td>0.00mL</td>
| |
| - | <td>1.95 mL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>10% APS</td>
| |
| - | <td>25 μL</td>
| |
| - | <td>25 μL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td>TEMED</td>
| |
| - | <td>2.5 μL</td>
| |
| - | <td>2.5 μL</td>
| |
| - | </tr>
| |
| - |
| |
| - | <tr>
| |
| - | <td><b>Total Volume</b></td>
| |
| - | <td><b>~5.0 mL</b></td>
| |
| - | <td><b>~3.0 mL</b></td>
| |
| - | </tr>
| |
| - | <table>
| |
| - |
| |
| - | <p>Note: Since our protein ladders don’t contain small molecular weight proteins, it is very useful to find a protein sample of a small protein to run as comparison on the gel.</p>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | </div>
| |
| - | </div>
| |
| - | </div>
| |
| - | </body>
| |
| - | </html>
| |
"
