Team:The Agency Escondido/notebook

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                 </ul>
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                 <a href="http://agency-stem.org"><img src="http://agency-stem.org/static/img/2013-heading-web.png" alt="Agency Logo"/></a>
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        <div id="sections">
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<ul>
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<ul id="nav">
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<li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido">About</a></li>
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    <li>
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<li id="selectedsection">Notebook</li>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido">About</a>
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<li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/safety">Safety</a></li>
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        <ul>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido">General</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/sandiego">San Diego</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/ourteam">Our Team</a></li>
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        </ul>
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    </li>
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<li>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project"><u>Project</u></a>
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        <ul>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/rock">Rock</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/paper">Paper</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/scissors">Scissors</a></li>
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        </ul>
 +
    </li>
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    <li>
 +
        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/notebook">Notebook</a>
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        <ul>
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            <li></li>
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        </ul>
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    </li>
 +
    <li>
 +
        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/safety">Safety</a>
 +
        <ul>
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            <li></li>
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        </ul>
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    </li>
</ul>
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          </div>
<h2>April 9</h2>
<h2>April 9</h2>
<p>Andrew Buss prepared 120 mL of LB broth</p>
<p>Andrew Buss prepared 120 mL of LB broth</p>
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<p>Vansh Singh transferred DNA of Part A, Part B, and NEB10 colonies to Ependorfs to find concentration of DNA</p>
<p>Vansh Singh transferred DNA of Part A, Part B, and NEB10 colonies to Ependorfs to find concentration of DNA</p>
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<p>Andrew Buss prepared 100 mL of LB broth using <a href="http://casp.agency-stem.org/media/G13-LP0003-14.pdf">revision 14 of our LB broth protocol</a>. However, autoclaving was deferred until a larger batch of material needed autoclaving.</p>
+
<p>Andrew Buss prepared 100 mL of LB broth using <a href="http://casp.agency-stem.org/media/G13-LP0003-14.pdf">revision 14 of our LB liquid protocol</a>. However, autoclaving was deferred until a larger batch of material needed autoclaving.</p>
<p>Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of ampicillin/kanamycin media (antibiotics to be ordered soon), these were cultured without antibiotics. </p>
<p>Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of ampicillin/kanamycin media (antibiotics to be ordered soon), these were cultured without antibiotics. </p>
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<p>Andrew Buss inoculated a second LB plate with a section of NEB10 cells from the 4/18 NEB10 plate.</p>
<p>Andrew Buss inoculated a second LB plate with a section of NEB10 cells from the 4/18 NEB10 plate.</p>
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</div>
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<h2>April 20 labeled media</h2>
<h2>April 20 labeled media</h2>
<table>
<table>
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<tr><td>Andrew Segina</td><td>LB plate</td><td>Part A from kit</td><td>Ampicillin, kanamycin</td><td></td></tr>
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<tr><td>Andrew Segina</td><td>LB plate</td><td>Part A from kit</td><td>Ampicillin, kanamycin</td><td>In refrigerator</td></tr>
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<tr><td>Andrew Segina</td><td>LB plate</td><td>Part B from kit</td><td>Ampicillin, kanamycin</td><td></td></tr>
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<tr><td>Andrew Segina</td><td>LB plate</td><td>Part B from kit</td><td>Ampicillin, kanamycin</td><td>In refrigerator</td></tr>
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<tr><td>Andrew Buss</td><td>SOB plate</td><td>NEB 10 from kit</td><td></td><td></td></tr>
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<tr><td>Andrew Buss</td><td>SOB plate</td><td>NEB 10 from kit</td><td></td><td>In refrigerator</td></tr>
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<tr><td>Andrew Segina</td><td>SOB plate</td><td>NEB 10 from kit</td><td></td><td></td></tr>
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<tr><td>Andrew Segina</td><td>SOB plate</td><td>NEB 10 from kit</td><td></td><td>In refrigerator</td></tr>
<tr><td>Andrew Buss</td><td>LB tube</td><td>Part A from kit</td><td></td><td>Incubating in dry bath</td></tr>
<tr><td>Andrew Buss</td><td>LB tube</td><td>Part A from kit</td><td></td><td>Incubating in dry bath</td></tr>
<tr><td>Andrew Buss</td><td>LB tube</td><td>Part B from kit</td><td></td><td>Incubating in dry bath</td></tr>
<tr><td>Andrew Buss</td><td>LB tube</td><td>Part B from kit</td><td></td><td>Incubating in dry bath</td></tr>
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<tr><td>Grant Hassinger</td><td>100mL LB broth</td><td></td></tr>
<tr><td>Grant Hassinger</td><td>100mL LB broth</td><td></td></tr>
</table>
</table>
 +
<h2>April 23</h2>
 +
<p>Andrew Buss's LB broth from April 17 was not autoclaved because there were no other autoclave operations with which to batch it. The broth was contaminated in the intervening time and is not usable. In the future, media will be autoclaved immediately after mixing.</p>
 +
<p>Vansh Singh performed DNA Ligation on the linearized plasmid Backbone of pSBCI3, and DNA parts of Part A and Part B according to <a href = "http://casp.agency-stem.org/media/G13-LP0008-17.pdf">revision 18 of Ligation</a>.</p>
 +
<p>Grant Hassinger reinoculated one LB tube with E. coli with parts B from Andrew Buss's </p>
 +
<p>Vansh Singh and Andrew Buss accompanied Andrew Segina to a local university to use a nanodrop machine to quantify the DNA samples from 4/18. The readings on all samples were outside of the range of the machine - either our samples were too concentrated or too dilute, or impurities were introduced. Supplies for agarose gel runs are on order - we will verify that we have any DNA fragments of the correct length when the supplies arrive.</p>
 +
<h2>April 24</h2>
 +
 +
<p>Our competent cells arrived from NEB. While we originally planned to begin transformation today, continued difficulties with the centrifuge occupied our efforts. One of six tubes was retained in dry ice inside a styrofoam ice box inside the -20 C freezer for transformation the next day - the other five were taken to a local university for storage at -80 C. </p>
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<p>Andrew Buss prepared LB broth according to <a href = "http://casp.agency-stem.org/media/G13-LP0003-14.pdf">revision 14 of our LB liquid protocol</a>.<p>
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<!-- each person below should write their own statements, including the amount of media produced. E.g. Grant J produced 150 mL, while Andrew prepared 100 mL -->
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<p >Vansh Singh, <!--<span style="text-decoration:line-through;">Evan Santos, Blaise Devalasco, Andrew Buss, and Grant Hassinger</span>--> prepared 40 uL LB broth according to <a href = "http://casp.agency-stem.org/media/G13-LP0003-32.pdf">revision 32 of LB liquid protocol</a>.<p>
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<h2>April 25</h2>
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<p>Ben Jones prepared 200 mL LB broth according to <a href = "http://casp.agency-stem.org/media/G13-LP0003-32.pdf">revision 32 of our LB liquid protocol</a>.<p>
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<p>Evan Santos prepared 300 mL LB broth according to <a href = "http://casp.agency-stem.org/media/G13-LP0003-32.pdf">revision 32 of LB liquid protocol</a>.</p>
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<p>Vansh Singh and Evan Santos researched promoters with different inducers for our project. Unfortunately, our search was unsuccessful because every inducible promoter we found was induced by arabinose. We will continue this search.</p>
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<p>Andrew Buss began transformation. Three 2.0 mL tubes were labeled and filled with the correct quantity of DNA. They were placed in the freezer when the protocol was aborted for lack of time. Tomorrow, we intend to complete transformation for the first time.</p>
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<h2>April 26</h2>
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<p>Ben Jones inoculated a new tube of LB from Andrew Buss's Part A from 4/17</p>
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<p>Andrew Buss completed transformation using the ligated products from 4/19. 50uL remaining from each sample has been replaced into the refrigerator.</p>
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<p>Later, Vansh Singh started transformation using the ligated products from 4/23 only to find Andrew Buss had used all but 50uL of the competent cells. Therefore Vansh Singh only completed transformation on the <b>Transformation Control</b> part. 50uL remaining from the <b>Transformation Control</b> part has been placed in the refrigerator.The LB agar plates with Transformation Control began incubation in 37 degrees Celsius at 7:30pm.<p>
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<h2>April 27</h2>
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<p>At 10:30 AM, Andrew Buss's Transformation Control plate displayed notably red colonies. However, while the Ligation Control plate displayed colonies, they did not appear red. The New Part plate did not appear to have been inoculated correctly as no colonies were evident. Vansh Singh's Transformation Control plate from the previous day included colonies, but they were not red.</p>
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<p>At 1:15 PM, the colonies on Andrew Buss's Ligation Control plate appeared noticeably red. The New Part plate still displayed no colonies. Andrew Buss reinoculated the plate using the remaining 50 uL of the New Part cell sample and spread the cells with glass beads. </p>
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<p>At 1:20 PM, Vansh Singh's Transformation control plate appeared noticeably red. He then inoculated a 5mL tube of Evan Santos's extra LB liquid from a red colony from the Transformation control plate.</p>
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<p>Vansh Singh produced another batch of LB liquid. His two previous tubes of E.Coli had been contaminated because the caps of the tubes came off in the autoclave. </p>
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<table>
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<thead>
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    <th colspan = "2">Transformation Results</th>
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</thead>
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<tbody>
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    <tr>
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          <td><solid>Vansh Singh Transformation Control</solid></td>
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          <td><img width=500px src = "https://static.igem.org/mediawiki/2013hs/6/6f/AgencyTransformationCTRL2013.jpg" id = "img"/></td>
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    </tr>
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    <tr>
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          <td><solid>Andrew Buss Ligation Control</solid></td>
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          <td><img width=500px src ="https://static.igem.org/mediawiki/2013hs/4/40/AgencyLBLigationCtrl2013.jpg" id = "img"/></td>
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    </tr>
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    <tr>
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          <td><solid>Andrew Buss Transformation Conrol</solid></td>
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          <td><img width=500px src ="https://static.igem.org/mediawiki/2013hs/9/93/AgencyRecombanationControl.jpg" id = "img"/></td>
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    </tr>
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</tbody>
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</table>
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<p>At 2:00 PM, closer examination revealed about 5 to 10 colonies sparsely scattered about the New Part plate that did not yet appear red. Their size suggested that they had been present before the second inoculation attempt. Therefore, the planned repetition of restriction and ligation operations was deferred until the colonies grow in size and number.</p>
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<p>Andrew Buss inoculated three tubes of LB broth (without antibiotics) with colonies from his New Part, Ligation Control, and Transformation Control plates. </p>
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<p>Andrew Buss performed restriction using Vansh Singh's miniprep products from 4/17, because the prepared DNA supplied in the kit for Part A, Part B, and the plasmid backbone, had been exhausted in the previous restriction step. According to Andrew Segina, the supplied NEB10 cells contained the plasmid backbone, so, while unfortunately not mentioned in Vansh's 4/17 log entry, they were run through miniprep process along with cells containing Parts A and B.</p>
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<p>As mentioned on 4/23, the concentration of these DNA samples could not be measured using a nanodrop machine, nor was there an opportunity to return to test more dilute samples. From the recorded miniprep yields of other teams, an extremely rough estimate of 75 ng/uL was assumed for later calculations: 6.66 uL of each DNA sample and 35.84 uL of distilled water per tube. No other reagent volumes were modified. No RFP control sample was used - the transformed ligation control plate demonstrated a successful ligation process.</p>
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<p>The lab closed at 6:00 PM before the thermocycler completed the program for restriction digest. The thermocycler program will hold the digested samples at 4 degrees C over the weekend until work is resumed on Monday. At the time of departure, the colonies on Andrew Buss's New Part plate did not appear distinctly red. </p>
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<h2>April 29</h2>
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<p>At 8:50 AM, Andrew Buss's New Part plate showed a pink lawn of bacteria. This indicates that the restriction, ligation, and transformation processes succeeded, albeit at low efficiency. Pictures forthcoming.</p>
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<p>Andrew Buss's LB tubes from 4/27 containing New Part and Ligation Control were of a pinkish hue, and bacteria that had adhered at the waterline to the inside surfaces of the tubes were strongly pink. The Transformation Control tube held clear LB and upon closer inspection there was no sign of the pipette tip that was cut for inoculation, so the media was not exposed to any bacteria.</p>
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<p>Andrew Buss ligated Parts A and B, plus the plasmid backbone digests, from the previous day. Transformation should occur tomorrow.</p>
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<p>Andrew Buss worked to improvise an agarose gel setup compatible with the Ready-To-Run Separation Unit. The unit was designed to operate with premade TBE/ethidium bromide agarose cassettes and without additional liquid running uffer. The first gel, attempted on Saturday, reported "E1": overcurrent. Draining the TAE buffer caused an "open circuit" error. The only arrangement with which the machine initially appeared electrically content was the gel contacting only narrow segments of the bare electrodes at a 30 degree angle away from normal, a setup entirely incompatible with practical use.</p>
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<p>The next idea was to cut the approximately 2.5"x4" gel into a 2.5"x2.5" square that would minimize electrode contact while keeping the charge gradient in the correct direction relative to the wells. This worked for a short while, but without TAE to maintain the gel, within a few minutes, the gel had shrunk enough to lose contact with the negative electrode. Even covering the gel surface with TAE again caused the "E1": over-current error. </p>
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<p>A second 1.5% agarose/TAE gel was cast in the unit, after sealing some nooks (the areas beyond the electrodes, for instance) with electrical tape. Andrew Segina suggested a more conservative 30 mL from the 50 mL used in the first gel, but this volume was insufficient to entirely fill the curved surface of the plate. An additional 30 mL was prepared and overlaid on the first without issue. However, this gel suffered effectively the same issues as the first: without TAE, an undercurrent, with TAE, an overcurrent. Periodically filling the widening gaps between the electrodes and the gaps with additional strips of agarose gel helped prolong operation.</p>
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<p>For testing, a sample of 2-log DNA ladder, a a sample of gel loading dye, and an opportune sample of 1% methylene blue (it was mentioned that it would stain DNA) were inserted into subsequent wells. The DNA ladder diffused quickly, the gel loading dye diffused more slowly and migrated in the correct direction, and the methylene blue migrated backwards to the negative electrode and began spilling into the unit's plate. Further research indicated that methylene blue held a positive charge and so migrated backwards. Andrew Buss discovered a protocol which revealed that methylene blue should be added after the gel run concludes. </p>
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<p>A third gel was cast in the unit from 50 mL of mix. The intention was to allow the gel to entirely fill the plate, including behind the electrodes. When the gel had cooled, these areas were cut out with a razor and disposed of. The gel was lightly doused in TAE, as were the spaces between the gel and the electrodes. In this arrangement, the unit continued for nearly 5 minutes before it reported an undercurrent condition again. </p>
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<p>Andrew Buss moved the gel to a mini gel box. The only available power supply had a range of 0-20V, so some previously inserted loading dye migrated very slowly. Tomorrow, it is hoped that we can borrow a benchtop power supply with a more reasonable output voltage range.</p>
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<p>Vansh Singh and Evan Santos searched for and found the promoters, inducers, and reporters for the project.<p>
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<p> Grant Hassinger started miniprep on his inoculated E. coli with parts A & B and attempted to pause the process through freezing at -20C</p>
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<h2>April 30</h2>
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<p>Andrew Buss obtained a 100/50 volt power supply designed specifically for electrophoresis. A gel loading dye was run alongside a DNA ladder for 50 minutes at 100 volts. The gel was then removed from the tray and soaked in 0.002% methylene blue at room temperature for 3 hours. Before the lab closed, the gel was left to wash in 1X TAE. </p>
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<p>After Vansh Singh completed plasmid design, Andrew Buss used the Parts Registry to identify existing parts that would eliminate later time-consuming steps for the "rock" plasmid. First, <a href="http://partsregistry.org/Part:BBa_S01022">an intermediate part</a> by Randy Rettberg eliminates the assembly of an RBS and a cyan fluorescent protein. <a href="http://partsregistry.org/Part:BBa_J37033">Another intermediate part</a> eliminates the assembly of an RBS and a LuxR coding region. These reduce the number of required assemblies to three, which cannot be parallelized because of the requirement below that we produce the independent AraC sensor.</p>
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<p>Andrew Buss documented the final and intermediate steps of assembly of the "rock" plasmid. First, we will construct <a href="http://partsregistry.org/Part:BBa_K1001750">a simple AraC sensor</a>. Then we will assemble this sensor with <a href="http://partsregistry.org/Part:BBa_J37033">a LuxR coding region</a> to form <a href="http://partsregistry.org/Part:BBa_K1001751">our final plasmid</a>. The intermediate AraC sensor will serve as a useful diagnostic tool, and will be submitted to the Registry as well.</p>
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<p>Andrew Buss began to write a high-level protocol for assembly, detailing the restriction and ligation steps required to complete the plasmid.</p>
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<p> Grant Hassinger resumed his miniprep but as the next step did not result in the desired effect, he concluded the cells should not be frozen </p>
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<h2>May 1</h2>
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<p>Andrew Buss's agarose gel was a uniform blue color, even after rinsing with TAE overnight. No bands were visible.</p>
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<p>Andrew Buss transformed competent cells with the ligation from 4/29.</p>
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<p>Andrew Buss resuspended DNA samples of BBa_R0080, BBa_B0010, BBa_J373033, and pSB1C3 from the Spring 2012 DNA distribution in 50 uL of distilled water each. Transformation is postponed until ampicillin media becomes available (ampicillin and other supplies are on order).</p>
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<h2>May 8</h2>
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<p>Andrew Buss's transformation plate from 5/1 showed no colonies. However, there was large black colony that appeared densest by the edge of the plate. Evidently, contamination was introduced during the transformation process, although the absence of other colonies suggests that the transformation process or a previous step did not work correctly.</p>
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<h2>May 9</h2>
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<p> Grant Jose inoculated lb with parts a and neb 10 </p>
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<p>Andrew Buss inoculated 5 mL of LB media with 100 uL of media containing NEB 10 cells. After inoculation, the spectrophotometer registered an OD 600 of .029. This did not vary over the next hour and the next three spectrophotometer readings. Andrew Segina hypothesized that this was a result of the (previously refrigerated) cells' slow return to a growth stage. The cells were left to incubate overnight.</p>
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<h2>May 10</h2>
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<p>Andrew Buss continued his spectrophotometry work from the previous night. Noticeable increase in OD 600 was observed - a range from .45 to .5. </p>
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<p>Andrew Buss tested a <a href="http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf">calcium chloride competent cell protocol</a>. Cell clumping presented obstacles to resuspension, so the test was simply to verify that the protocol did not entirely kill NEB 10 cells.</p>
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<p>Ben Jones ran a miniprep on Evan Santos's 3A Construction Protocol Part A</p>
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<p> Evan Santos ran a miniprep on Grant Jose's 5/9/13 culture of 3A Construction Protocol Part B</p>
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<h2>May 15</h2>
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<p>Evan Santos inoculated 600 mL of LB media with 25 uL of media containing NEB-10 cells.</p>
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<h2>May 21</h2>
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<p>Andrew Segina made 1mL 1000x stock solutions of Ampicillin, Kanamycin Sulfate, and Chloramphenicol, as per the protocol found in CASP.</p>
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<p>Grant Hassinger and Blaise DeValasco did a run of transforming Cells with I13500 and E0040 but the label wiped off and they became indistinguishable so the work was bleached and disposed of.</p>
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<h2>May 30</h2>
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<p>Evan Santos transformed competent cells for parts BBa_R0062 and BBa_I13500.</p>
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Latest revision as of 23:15, 21 June 2013