Team:Lethbridge Canada/notebook june

From 2013hs.igem.org

(Difference between revisions)
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<div id="mainContent">
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Stuff
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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>
 +
<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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<li><a href="notebook_may.html#entry_twenty">May 17, 2013: Restrictions and Gel</a></li>
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<li><a href="notebook_may.html#entry_twenty_one">May 22, 2013: Ligation</a></li>
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<li><a href="notebook_may.html#entry_twenty_two">May 22, 2013: Transformations</a></li>
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<li><a href="notebook_may.html#entry_twenty_three">May 23, 2013: Transformations</a></li>
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<li><a href="notebook_may.html#entry_twenty_four">May 23, 2013: Picked Oxytocin and Transformations</a></li>
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<li><a href="notebook_may.html#entry_twenty_five">May 24, 2013: Glycerol Stock</a></li>
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<li><a href="notebook_may.html#entry_twenty_six">May 24, 2013: Miniprep</a></li>
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<li><a href="notebook_may.html#entry_twenty_seven">May 26, 2013: Miniprep</a></li>
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<li><a href="notebook_may.html#entry_twenty_eight">May 26, 2013: Restriction Digestion</a></li>
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<li><a href="notebook_may.html#entry_twenty_nine">May 26, 2013: Agarose gel of Digestion</a></li>
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<li><a href="notebook_may.html#entry_thirty">May 26, 2013: Digestion Protocol</a></li>
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<li><a href="notebook_may.html#entry_thirty_one">May 31, 2013: Agarose Gel</a></li>
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<li><a href="notebook_may.html#entry_thirty_two">May 31, 2013: Everything</a></li>
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<li><a href="notebook_may.html#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="notebook_june.html">June</a></li>
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<li><a href="notebook_june.html#entry_one">June 1, 2013: Restrictions</a></li>
 +
<li><a href="notebook_june.html#entry_two">June 2, 2013: Transformation</a></li>
 +
<li><a href="notebook_june.html#entry_three">June 4, 2013: Picked Colonies</a></li>
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<li><a href="notebook_june.html#entry_four">June 10, 2013: Sent constructs for sequencing</a></li>
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<li><a href="notebook_june.html#entry_five">Transformation of OXY into BL21</a></li>
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<li><a href="notebook_june.html#entry_six">Restricted and Ligated and Transformed</a></li>
 +
<li><a href="notebook_june.html#entry_seven">Picked Cells</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">Restrictions</h1>
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<ul class="notebook_entry_list">
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<li>Resticted(total 60 ul dna) K314100 in psb1c3 with spe1 and Pst1</li>
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<li>Resticted(total 60ul dna) Oxytocin in Puc19a with Xba1 and Pst1</li>
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<li>Resticted(total 60ul dna) NEC1puc19a with Xba1 and Pst1</li>
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<li>Set 12ul aside from each reaction and gel extracted(GEX) the remaining  48ul</li>
 +
<li>Combined heat killed restiction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80</li>
 +
<li>K314100 – psb1c3 (s,p), with: Oxytocin (x,p)</li>
 +
<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 20ul high efficiency Dh5a cells and 50ul lab made competent Dh5a</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>
 +
<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 20ul non gel extracted Dh5a 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="leth_hs_2013_gel_extraction_06_02_2013_2.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 – minipreped and restricted all colonies picked so far at E,P 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 nec 1</li>
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<li><img src="leth_hs_2013_gel_extraction_06_02_2013_1.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">
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<li>Date: June 10, 2013</li>
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<li class="notebook_nested_start">Sent 10ul DNA with 5ul of 5uM 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 neurophysin1 </li>
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</ul>
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</li>
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<li class="notebook_nested_start">June 12th results arrived</li>
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<li><ul class="notebook_nested_list">
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<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>
 +
<li>J23113-J06702- positive for J06702 but no promoter found</li>
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<li>K314100-oxytocin neurophysin1 – Positive results full sequence found</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">
 +
<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 minipreped plasmid DNA of K314100 –Oxytocin Neurophysin1  in psb1c3 into 15 ul of Bl21 E.coli cells </li>
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<li>Let grow over night at 37 on CAM plates</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">
 +
<div id="entry_six">
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<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 followed by 20 min heat kill at 80</li>
 +
<li>Mixed restrictions in ratio of 10ul nec1 and 2ul k314100 in psb1c3  let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80</li>
 +
<li>Transformed 2ul into 20ul of dh5a ecoli highly compentent cells, incubated overnight at 37</li>
 +
</ul>
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<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 in psb1c3 grew in Bl21 from lastnights tranformation, picked 4 colonies and grew up in 5ml of LB overnight at 37 with shaking</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>
</div>
</div>

Revision as of 01:28, 17 June 2013

Restrictions

  • Resticted(total 60 ul dna) K314100 in psb1c3 with spe1 and Pst1
  • Resticted(total 60ul dna) Oxytocin in Puc19a with Xba1 and Pst1
  • Resticted(total 60ul dna) NEC1puc19a with Xba1 and Pst1
  • Set 12ul aside from each reaction and gel extracted(GEX) the remaining 48ul
  • Combined heat killed restiction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80
  • 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)

Back to Top

Transformation

  • Transformed ligations into 20ul high efficiency Dh5a cells and 50ul lab made competent Dh5a
    • 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 20ul non gel extracted Dh5a E. coli high efficiency cells grew

Back to Top

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 – minipreped and restricted all colonies picked so far at E,P 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 nec 1
  • Gel on June 4, 2013

Back to Top

Picked Colonies

  • Date: June 10, 2013
  • Sent 10ul DNA with 5ul of 5uM vf2 primer to genewize
    • j23100-J06702
    • J23108-j06702
    • J23113-J06702
    • K314100-oxytocin neurophysin1
  • 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 neurophysin1 – Positive results full sequence found

Back to Top

Transformation of OXY into BL21

  • Date: June 13, 2013
  • Transform minipreped plasmid DNA of K314100 –Oxytocin Neurophysin1 in psb1c3 into 15 ul of Bl21 E.coli cells
  • Let grow over night at 37 on CAM plates

Back to Top

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 followed by 20 min heat kill at 80
  • Mixed restrictions in ratio of 10ul nec1 and 2ul k314100 in psb1c3 let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80
  • Transformed 2ul into 20ul of dh5a ecoli highly compentent cells, incubated overnight at 37

Back to Top

Picked Cells

  • Date: June 13, 2013
  • K314100 – Oxytocin neurophysin in psb1c3 grew in Bl21 from lastnights tranformation, picked 4 colonies and grew up in 5ml of LB overnight at 37 with shaking

Back to Top

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