Team:Linkoping Sweden/Notes

From 2014.igem.org

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<img src="https://static.igem.org/mediawiki/2014/a/a9/Linkoping_sweden_notebook.jpg" width="1100px" title="Notebook"/>
<img src="https://static.igem.org/mediawiki/2014/a/a9/Linkoping_sweden_notebook.jpg" width="1100px" title="Notebook"/>
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<div id="june" class="text-panel">
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<h2 class="clickable ">June</h2>
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<h3>June 19th</h3>
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<p>Supercompetent E-Coli was given to us from one of our professors. We tested the health of the bacteria by letting them grow on a control plate at 37 ºC.</p>
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<h3>June 24th</h3>
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<p>Transformation of RFP control (10pg/ul) plasmid into supercompetent cells by heat shock method.</p>
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<h3>June 25th</h3>
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<p>Did the transformation of RFP control again since the first transformation went wrong.</p>
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<h3>June 26th</h3>
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<p>RFP control plate looked good this time.</p>
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<h3>June 30th</h3>
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<p>Heat shock transformation of:<br>His-TEV-protein plasmid (provided by professor LG Mårtensson)<br>BBa_K525998 (promotor+RBS)<br>RFP PSB1C3-J06504</p>
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<h2 class="clickable ">July</h2>
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<h3>July 1st</h3>
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<p>Re-streak of:<br>His-TEV-protein plasmid (provided by professor LG Mårtensson), clone 1-2<br>BBa_K525998 (promotor+RBS), clone 1-4</p>
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<p>New transformation of:<br>RFP PSB1C3-J06504 again since the bacterias did not grow.</p>
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<h3>July 2nd</h3>
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<p>Re-streak of:<br>RFP PSB1C3-J06504</p>
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<p>Transformation of:<br>RFP-control with electrocompetent cells</p>
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<p>Over-night of:<br>PSB1C3 – T7 promotor<br>His-TEV-protein</p>
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<p>Preparation of starter:<br>10 colonies of transformed His-TEV-protein were used. Induction over-night.</p>
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<h3>July 3rd</h3>
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<p>Plasmid preparation of:<br>PSB1C3-T7 promoter, clone 1-3<br>His-TEV-protein, clone 1, 2</p>
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<p>Over-night on:<br>PSB1C3-J06504-RFP</p>
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<p>Re-streak of:<br>RFP-control</p>
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<p>Preparation of:<br>Large culture of transformed His-TEV-protein.</p>
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<p>Measurement of OD600 and induction with IPTG over-night.</p>
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<h3>July 4th</h3>
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<p>Plasmid preparation of:<br>J06504-RFP, clone 1, 2</p>
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<p>Transformation of:<br>PSB1C3-E1010-RFP</p>
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<p>Competency control of:<br>Electro competent cells</p>
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<p>Harvest of large culture by centrifugation followed by lysation of bacterial cells through sonication.</p>
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<p>Purification of:<br>His-TEV-protein (lysate from sonication) by the use of nickel-column.</p>
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<h3>July 5th</h3>
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<p>Re-streak of: PSB1C3-E1010-RFP, clones 1-6</p>
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<h3>July 6th</h3>
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<p>Over-night of: PSB1C3-E1010-RFP, clones 1-6</p>
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<h3>July 7th</h3>
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<p>Plasmid preparation of:<br>PSB1C3-E1010-RFP overnight-culture, clone 5</p>
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<h3>July 8th</h3>
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<p>Analysis of protein content in:<br>Fractions from nickel-column purification by the use of Bradford assay.<br>Fractions containing protein was analyzed by the use of SDS-page. </p>
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<h3>July 9th</h3>
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<p>Restriction digest performed by spin-kit and the use of Xba1 and Spe1 followed by ligation and transformation (electroporation). </p>
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<h3>July 10th</h3>
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<p>Fractions containing His-TEV-protein was set on TEV-cleavage over-night.</p>
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<h3>July 11th</h3>
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<p>Reversed nickel-column purification of:<br>His-TEV-protein after TEV cleavage.<br>Fractions from the reversed nickel-column purification as well as a fraction both before and after TEV cleavage was controlled by SDS-page.</p>
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<h3>July 15th</h3>
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<p>Restriction digest test on all restriction enzymes (except FD PstI) and put on agarose elektrofores gel. (EcoRI, XbaI, SpeI, PstI)</p>
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<p>Nanodrop of PSB1C3-E1010 and His-Tev to control the purity and concentration.</p>
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<h3>July 16th</h3>
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<p>Analyze of the agarose gel including staining with EtBr, destaining with water and visualization under UV-light.</p>
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<h3>July 21st</h3>
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<p>New fransformation of PSB1C3-E1010 and His-Tev scince the concentrations from previous plasmid preps were too low.</p>
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<h3>July 29th</h3>
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<p>O/N on His-TEV-protein, clones 1-4.</p>
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