Difference between revisions of "Team:Cadets2Vets/Demonstrate"

On July 25, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace ran a second ticket assay to increase the power of the previous test and provide clearer results.

To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.

The reaction solution contained 7.5 µL S30 Extract, 10 µL S30 Promega, 2.5 µL amino (+), 0.5 µL amino (-), and 2 µL molecular water. For the positive test, 2.5 µL of our circuit plasmid and machinery was added, and the 2 µL molecular water contained arsenite and arsenate at a 150 µM concentration. The negative control ticket used only 2 µL of reaction mixture for the first two columns of wells on the left, and the other two columns of wells were filled with 2 µL of molecular water. The positive test ticket used 2 µL of the test mixture in the first column of wells, and 4 µL of test mixture in the second column of wells. The other two columns of wells were filled with 2 µL of molecular water.

Fig 4.1 and Fig 4.2 - The Positive Test Circuit (top) with first row containing 2 µL of test mixture, and the second row containing 4 µL of test mixture. Possible dose response is visible. The Negative Control Circuit (bottom), containing 2 µL of reaction mixture in first and second columns.

These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1 and Fig 1.2). Once again, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first and second columns of wells, and qualitatively it appears that there may have been some form of dose response, as the second column of wells appears brighter. The negative control appears to have dried again slightly, and the Cadets2Vets team will be aliquoting more reaction mixture into each well during later tests to try and combat this issue.