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<p dir="ltr">Notes:</p><p dir="ltr">Based off of the recommendations from previous report, it was determined that the two primary needs were to identify a means of quantifying GFP production, and QC/QA the plasmid.</p><br><p dir="ltr">Proposed route:</p><ul><li dir="ltr"><p dir="ltr">Attempt to see if LC 480 II is capable of monitoring GFP production</p></li><li dir="ltr"><p dir="ltr">QC/QA Plasmid:</p><ul><li dir="ltr"><p dir="ltr">Plasmid Gel</p></li><li dir="ltr"><p dir="ltr">M13 amplification</p></li></ul></li><li dir="ltr"><p dir="ltr">Attempt to quantify the culture, at 25 µM of arsenite and arsenate</p></li></ul><p><br></p><p dir="ltr">Experiments:</p><ul><li dir="ltr"><p dir="ltr">Validate the use of LC 480 II utilizing T7 constitutive GFP construct</p><ul><li dir="ltr"><p dir="ltr">Experiment was run in triplicate with both</p><ul><li dir="ltr"><p dir="ltr">Controls: T7 expression blank, Solution blank (water), and Plasmid blank</p></li></ul></li></ul></li><li dir="ltr"><p dir="ltr">QC/QA of Ars 1.1 plasmid circuit</p><ul><li dir="ltr"><p dir="ltr">Minipreps were done in triplicate and tested on the nanodrop</p></li><li dir="ltr"><p dir="ltr">The triplicate minipreps were run on three separate gels at 0.7% agarose</p></li><li dir="ltr"><p dir="ltr">All three mini-preps were tested with an M13 cloning site amplification and the insert size was checked on a 1% agarose gel</p></li></ul></li><li dir="ltr"><p dir="ltr">Culture of Ars 1.1 transformed E. coli were exposed to 25uM Arsenate+Arsenite at 37C in the LC 480 II for 24 hours</p><ul><li dir="ltr"><p dir="ltr">Percentages are in m/V</p><ul><li dir="ltr"><p dir="ltr">LB 4%</p></li><li dir="ltr"><p dir="ltr">Ampicillin 0.01%</p></li></ul></li></ul></li></ul><p><br><br></p><p dir="ltr">Results:</p><p dir="ltr">Validate the use of LC 480 II utilizing T7 constitutive GFP construct- Successful</p><br><p dir="ltr"><span id="docs-internal-guid-2e42da7c-1809-2e74-a25d-cd92979219b5"><span style="font-size: 11pt; font-family: Calibri; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><img data-cke-saved-src="https://lh6.googleusercontent.com/1tDorZN6nqf1f5DpZM7tLIIUXMWgCKoghwLUWqq-cq4RVDOiAjHX0-GUlix8vBHtooh1O75dYNjnYLKn-3shpK91Wg_yBexxtOYdzmgdXWTo7Sko5GYk0BEB3kh7Jd-hZSslNfNTzLXzP4m-pg" src="https://lh6.googleusercontent.com/1tDorZN6nqf1f5DpZM7tLIIUXMWgCKoghwLUWqq-cq4RVDOiAjHX0-GUlix8vBHtooh1O75dYNjnYLKn-3shpK91Wg_yBexxtOYdzmgdXWTo7Sko5GYk0BEB3kh7Jd-hZSslNfNTzLXzP4m-pg" width="635" height="278" style="border: none; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" alt="C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IMG_20170628_133008 (1).jpg"></span></span><br></p><p dir="ltr">The LC 480 II was successfully able to monitor the increase of GFP over the incubation time.</p><p><br><br><br></p><p dir="ltr">QC/QA the Plasmid</p><p dir="ltr">Circularized plasmid gels proved inconclusive, however M13 amplification did provide strong indication of the proper fragment. The fragment size is 1461 bp, with the second bright band in the ladder being equal to 1650 bp. All minipreps exhibit a band slightly below the 1625 bp band of the ladder, which is evident of the proper fragment size.</p><p style="text-align: center;"><br><span id="docs-internal-guid-2e42da7c-1809-9bd9-a8ce-7360bc5c67db"><img data-cke-saved-src="https://lh3.googleusercontent.com/6MruaZWKeTSh4V1jEiRrlkapQG7aHHYbEAxN7qGEJ4eQxruKQkbaPAnOXFW0EX3EMy7GG5QyMXAV1n6m0vyJzxDCU7fiAtd4va4ZaiMLR38crAZYLT25LVGz5RABdcarBGxWjgvzCCOwFuvE7w" src="https://lh3.googleusercontent.com/6MruaZWKeTSh4V1jEiRrlkapQG7aHHYbEAxN7qGEJ4eQxruKQkbaPAnOXFW0EX3EMy7GG5QyMXAV1n6m0vyJzxDCU7fiAtd4va4ZaiMLR38crAZYLT25LVGz5RABdcarBGxWjgvzCCOwFuvE7w" width="520" height="605" style="border: none; transform: rotate(0.00rad); -webkit-transform: rotate(0.00rad);" alt="C:\Users\Ryan\AppData\Local\Microsoft\Windows\INetCache\Content.Word\IGEM M13 GEL IN TRIPLICATE 07032017.jpg"></span><br><br><img data-cke-saved-src="https://docs.google.com/drawings/d/s1v4_DxEHt4ufxIOzmjoXKw/image?w=219&amp;h=148&amp;rev=1&amp;ac=1" src="https://docs.google.com/drawings/d/s1v4_DxEHt4ufxIOzmjoXKw/image?w=219&amp;h=148&amp;rev=1&amp;ac=1"></p><div><br></div>

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<h2 id="element-1405b4d3d7f8829" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu  allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >During the initial test of the circuit in the presence of 25 um arsenite and arsenate solution, we were able to generate a fluorescent signal using the Light Cycler. In the legend above, Negative Control 1 is actually Positive Control 3.<br></h2>