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| − | {{Elan_Vital_Korea/Header}}
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| − | <html>
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| − | <body>
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| − | <div class="sub-top">
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| − | <img src="https://static.igem.org/mediawiki/2015/d/d9/Sy_notebook-top.jpg" />
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| − | </div>
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| − | <div class="list-note-01">
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| − | <h1 class="day">4/28/15</h1>
| + | |
| − | <p class="content-txt">We received the DNA distribution kit from iGEM, and in order to amplify our amount of DNA, we transformed the DNA in competent cells, using the following LB growth medium.</p>
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| − | <h2 class="sub-title">1. Solid Medium</h2>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="240px" />
| + | |
| − | <col width="80px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>Trypton</th>
| + | |
| − | <td class="back-y">2g</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Sodium chloride</th>
| + | |
| − | <td class="back-y">2g</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Yeast</th>
| + | |
| − | <td class="back-y">1g</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">BactoAgar</th>
| + | |
| − | <td class="bdr-bt-none back-y">3g</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <h3 class="cmt">*D.W 200ml standard</h3>
| + | |
| − | <h2 class="sub-title">2. Liquid Medium</h2>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="240px" />
| + | |
| − | <col width="80px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>Trypton</th>
| + | |
| − | <td class="back-y">2g</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Sodium chloride</th>
| + | |
| − | <td class="back-y">2g</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">Yeast</th>
| + | |
| − | <td class="bdr-bt-none back-y">1g</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <h3 class="cmt">*D.W 200ml standard</h3>
| + | |
| − | <p class="content-txt">Because we needed to add antibiotics, we created a stock solution, with a concentration of 34mg/ml, diluted by 1000 times. When the growth medium cooled, we put in antibiotics.<br /><br />
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| − | Next, we marked the plasmids that we wanted to use from the DNA distribution kit.<br />
| + | |
| − | Then we added 10ul of distilled water to each plasmid, and stirred well, which caused the plasmid to turn red. We left the mixture to lay for 5 minutes.<br /><br />
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| − | These were the plasmids we had selected:</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="220px" />
| + | |
| − | <col width="265px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>BBa J23100</th>
| + | |
| − | <td class="left">17D plate 4 (ampicillin)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa B0030</th>
| + | |
| − | <td class="left">4G plate 4</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa C0062</th>
| + | |
| − | <td class="left">4L plate 2</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa B0024</th>
| + | |
| − | <td class="left">1D plate 4</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa R0062</th>
| + | |
| − | <td class="left">6H plate 2</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa E0040</th>
| + | |
| − | <td class="left">13L plate 4 (ampicillin)</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa B0015</th>
| + | |
| − | <td class="left">3F plate 3</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>BBa C0060</th>
| + | |
| − | <td class="left">4H plate 2</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">BBa I732006</th>
| + | |
| − | <td class="bdr-bt-none left">3B plate 3</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <p class="content-txt">Next, we prepared the transformation of the plasmids into competent cells. Since the cells had been stored at 70°C, we had to leave them out previous to the experiment to let them thaw, along with the S.O.C medium.<br /><br />
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| − | First, we added 3ul of DNA to the cells, and shook the mixture (pipetting didn’t do the job). After leaving the mixture on ice for 30 minutes, we heatshocked it at 42°C, for 30 seconds. Then, we left it on ice again for two minutes.<br /><br />
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| − | After that, we added 250ul of a prewarmed S.O.C medium, and left the mixture in a shaking incubator at 37°C and 225rpm for an hour. We then spread the mixture onto an LB plate, and allowed it to incubate overnight, for 16 hours.</p>
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| − | <h1 class="day m40">4/29/15</h1>
| + | |
| − | <p class="content-txt">The previous results were divided, as B0030, C0062, R0062, I732006, B0015 formed observable colonies, while J23100, B0024, C0060, E0040 did not. Thus, we decided to re-spread these four onto another LB plate.<br /><br />Successful Results:</p>
| + | |
| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/5/5e/Sy_note-img-1.jpg" /></p>
| + | |
| − | <h1 class="day m40">4/30/15</h1>
| + | |
| − | <p class="content-txt">We retransformed J23100, B0024, C0060, and E0040, and spread them onto a growth medium with ampicillin.</p>
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| − | <h1 class="day m40">5/1/15</h1>
| + | |
| − | <p class="content-txt">As of May 1>st C0060, E0040 colony were additionally verified, while B0024, J23100 still continued to not show any sign of colony growth. We decided to run a miniprep with the successful colonies B0030, C0062, R0062, I732006, B0015, C0060, and E0040, and thus left them in a 5ml liquid growth medium each.<br /><br />Successful Results:</p>
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| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/a/a4/Sy_note-img-2.jpg" /></p>
| + | |
| − | <h1 class="day m40">5/2/15</h1>
| + | |
| − | <p class="content-txt">We ran the miniprep, which was unsuccessful as the resulting amount of DNA was too small to work with.</p>
| + | |
| − | <h1 class="day m40">5/4/15</h1>
| + | |
| − | <p class="content-txt">Two weeks after receiving the DNA distribution kit from iGEM, we got the plasmids we needed to work with two weeks later, in bacterial cells.</p>
| + | |
| − | <h1 class="day m40">5/6/15</h1>
| + | |
| − | <p class="content-txt">We used the successful colonies to run our experiments. We transformed B0024 into a competent cell and spread it onto an ampicillin growth medium. We streaked bacteria containing J23100 and I732073. We also prepared C0060 and B0030 for a miniprep, and put them each in a 5ml liquid growth medium.</p>
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| − | <h1 class="day m40">5/7/15</h1>
| + | |
| − | <p class="content-txt">We looked at the results of the procedures of the day before, and while B0024 had no observable colony growth, J23100 and I732073 did. Then, we ran a miniprep on C0060 and B0030, which we had prepared for 18 hours the day before.</p>
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| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/4/46/Sy_note-img-3.jpg" /></p>
| + | |
| − | <h1 class="day m40">5/10/15</h1>
| + | |
| − | <p class="content-txt">The team made the decision to change the plasmids we were working with, opting for the J37032 plasmid, a LuxRresponsive GFP, in order to save time. We also changed our terminator to the K823017 plasmid. B0024 was supposed to be our bidirectional terminator, but because it couldn’t produce enough DNA, we had to find another plasmid to replace it.<br /><br />Here is the complete list of plasmids we are using:</p>
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| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="250px" />
| + | |
| − | <col width="250px" />
| + | |
| − | <col width="250px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>constitutive promoter</th>
| + | |
| − | <td class="left bdr-right">J23100</td>
| + | |
| − | <td class="left">ampicillin</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>RBS</th>
| + | |
| − | <td class="left bdr-right">B0030</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>LuxR</th>
| + | |
| − | <td class="left bdr-right">C0062</td>
| + | |
| − | <td class="left">ampicillin</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>terminator</th>
| + | |
| − | <td class="left bdr-right">K823017</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>LuxRresponsive GFP</th>
| + | |
| − | <td class="left bdr-right">J37032</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>terminator</th>
| + | |
| − | <td class="left bdr-right">B0015</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">AiiA</th>
| + | |
| − | <td class="left bdr-right bdr-bt-none">C0060</td>
| + | |
| − | <td class="left bdr-bt-none">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <h2 class="sub-title">1. LuxR</h2>
| + | |
| − | <p class="content-txt m-10">C0062 (vector) : EcoRI XbaI<br />B0030, J23100 (fragment) : EcoRI SpeI</p>
| + | |
| − | <h2 class="sub-title">2. GFP</h2>
| + | |
| − | <p class="content-txt m-10">J37032</p>
| + | |
| − | <h2 class="sub-title">3. LuxRGFP</h2>
| + | |
| − | <p class="content-txt m-10">K823017 (fragment) : EcoRI SpeI<br />promoter RBSLuxR (vector) : EcoRI XbaI<br />J37032 (fragment) : EcoRI SpeI</p>
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| − | <h2 class="sub-title">4. Test</h2>
| + | |
| − | <p class="content-txt m-10">C0060 (vector) : EcoRI XbaII<br />B0030, J23100 (fragment) : EcoRI SpeI</p>
| + | |
| − | <h1 class="day m40">5/11/15</h1>
| + | |
| − | <p class="content-txt">We decided to make 1% concentration agarose gel, using 0.7 grams of Seakem LE Agarose, 0.3 grams of Nusieye GTG agarose, and 100 mililliters of 1xTBE buffer. After putting all the mentioned ingredients into a flask, we put wrap over the entrance, and microwaved for two minutes, melting it entirely. Then we solidified the agarose by pouring it into a mold. Out of the DNA kit, we also transformed K823017 and J37032 into competent cells.</p>
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| − | <h1 class="day m40">5/11/15</h1>
| + | |
| − | <p class="content-txt">We verified the transformations from the day before, and both the K823017 and the J37032 colony were successful.</p>
| + | |
| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/3/3a/Sy_note-img-4.jpg" /></p>
| + | |
| − | <h1 class="day m40">5/13/15~5/19/15</h1>
| + | |
| − | <p class="content-txt">In order to increase the amount of plasmids of B0015, B0030, J37032, K823017, C0060, C0062, I732073, and J23100, we did minipreps and midipreps, which left us with more viable DNA to work with.</p>
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| − | <h1 class="day m40">5/20/15</h1>
| + | |
| − | <p class="content-txt">To verify whether the plasmids we’ve created were relevant to our experiment, we cut them using a restriction enzyme.<br /><br />These were the plasmids we used: B0015, B0030, J37032, K823017, C0060, C0062, I732073, J23100<br /><br />The results (depending on the enzyme used, highlighted in bold):.</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="240px" />
| + | |
| − | <col width="80px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>DNA of each</th>
| + | |
| − | <td class="back-y">1uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>XbaI/PstI</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Buffer</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Distilled Water</th>
| + | |
| − | <td class="back-y">15uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">Total</th>
| + | |
| − | <td class="bdr-bt-none back-y">20uL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <h2 class="sub-title align-c">XbaI (8) ↰↱ PstI (8)<br /><img src="https://static.igem.org/mediawiki/2015/3/3d/Sy_note-img-5.jpg" /></h2>
| + | |
| − | <p class="content-txt">This was the order of the gel electrophoresis markers:<br /><br />
| + | |
| − | (2.19kb, 2.2kb, 2.08kb, 3.0kb, 2.88kb, 2.85kb, 5.1kb, 2.1kb)<br />
| + | |
| − | B0015, K823017, B0030, J37032, C0060, C0062, I732073, J23100 / XbaI<br />
| + | |
| − | B0015, K823017, B0030, J37032, C0060, C0062, I732073, J23100 / PstI<br /><br />
| + | |
| − | C0062 and J23100 had larger sizes than were expected.</p>
| + | |
| − | <h1 class="day m40">5/29/15</h1>
| + | |
| − | <p class="content-txt">Due to our electrophoresis results being incorrect, in order to verify the existence of fragments we had to do a double digestion procedure, cutting B0015, K823017, B0030, J37032, C0060, I732073, J23100 with EcoRI and XbaI.</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="240px" />
| + | |
| − | <col width="80px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>DNA of each</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>EcoRI</th>
| + | |
| − | <td class="back-y">1uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>PstI</th>
| + | |
| − | <td class="back-y">1uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Buffer</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Distilled Water</th>
| + | |
| − | <td class="back-y">14uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">Total</th>
| + | |
| − | <td class="bdr-bt-none back-y">20uL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <p class="content-txt">Because of C0062 being far too big in the electrophoresis results, we redid the miniprep.<br /><br />
| + | |
| − | Since we got two types of C0062 from the iGEM kit, one that grows in an ampicillin medium and one that grows in a chloramphenicol medium. We decided to do a miniprep, then run a restriction enzyme procedure to see which one would be better for our experiment. We then prepared the C0062s for a miniprep.</p>
| + | |
| − | <h1 class="day m40">6/1/15</h1>
| + | |
| − | <p class="content-txt">We ran a miniprep on C0062.<br />C0062 #1, #2, #3 (ampicillin)<br />C0062 #1, #2, #3, #4, #5 ( chloramphenicol )</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="240px" />
| + | |
| − | <col width="80px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>DNA of each</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>EcoRI</th>
| + | |
| − | <td class="back-y">1uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>PstI</th>
| + | |
| − | <td class="back-y">1uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Buffer</th>
| + | |
| − | <td class="back-y">2uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>Distilled Water</th>
| + | |
| − | <td class="back-y">14uL</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">Total</th>
| + | |
| − | <td class="bdr-bt-none back-y">20uL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/b/b0/Sy_note-img-6.jpg" /></p>
| + | |
| − | <p class="content-txt">All plasmids were cut with EcoRI and PstI, then underwent gel electrophoresis. Looking at the DNA map of J23100, which had 2,105bp, we noticed that when we cut with EcoRI and PstI, there were two fragments, of 2kbp and 850bp.</p>
| + | |
| − | <p class="img-align"><img src="https://static.igem.org/mediawiki/2015/f/f3/Sy_note-img-7.jpg" /></p>
| + | |
| − | <p class="content-txt txt-i">*If we cut C0062 with EcoRI and PstI, we get two fragments as well, of 2,030 bp and 822bp.<br />The plasmids labeled ① ② ③ have ampicillin resistance vectors.<br />The plasmids labeled ④ ⑤ ⑥ ⑦ ⑧ have chloramphenicol resistance vectors. Plasmid ⑧ is a C0062.</p>
| + | |
| − | <p class="content-txt">While the DNA map states that the sizes of plasmids 1, 2, 3 are accurate, because we know that they ampicillin resistance genes in the vector, there are issues with our experiment scheme.<br /><br />To be able to verify the above results, we ran sequencing with B0030, C0062, and J23100.<br /><br />These were the sequencing primers:</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="80px" />
| + | |
| − | <col width="300px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>VF2</th>
| + | |
| − | <td class="left">TGC CAC CTG ACG TCT AAG AA</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">VR</th>
| + | |
| − | <td class="bdr-bt-none left">ATT ACC GCC TTT GAG TGA GC</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <h1 class="day m40">6/5/15 ~ 6/7/15</h1>
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| − | <p class="content-txt">We sent J23100, B0030, C0062, C0060, B0015, K823017, J37032, and J61100 plasmid to a sequencing company to see thee exact DNA sequence of our samples.<br /><br />Out of these, J23100, B0030, and B0015 had different sequence results than we had expected when comparing with samples on the Registry.<br /><br />So we decided to change these parts.</p>
| + | |
| − | <div class="tbl">
| + | |
| − | <table cellpadding="0" cellspacing="0">
| + | |
| − | <colgroup>
| + | |
| − | <col width="250px" />
| + | |
| − | <col width="250px" />
| + | |
| − | <col width="250px" />
| + | |
| − | </colgroup>
| + | |
| − | <tr>
| + | |
| − | <th>constitutive promoter</th>
| + | |
| − | <td class="left bdr-right"><font class="txt-g">J23100</font> -> J61100</td>
| + | |
| − | <td class="left">ampicillin</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>RBS</th>
| + | |
| − | <td class="left bdr-right"><font class="txt-g">B0030</font> -> J61100</td>
| + | |
| − | <td class="left"></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>LuxR</th>
| + | |
| − | <td class="left bdr-right">C0062</td>
| + | |
| − | <td class="left">ampicillin</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>terminator</th>
| + | |
| − | <td class="left bdr-right"><font class="txt-g">K823017</font> -> K823017</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th>LuxRresponsive GFP</th>
| + | |
| − | <td class="left bdr-right">J37032</td>
| + | |
| − | <td class="left">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <th class="bdr-bt-none">AiiA</th>
| + | |
| − | <td class="left bdr-right bdr-bt-none">C0060</td>
| + | |
| − | <td class="left bdr-bt-none">chloramphenicol</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <p class="content-txt">BBa J61100 plate 4 16C<br />BBa C0062 plate 2 4L<br />BBa K823017 plate 1 3D<br />BBa J37032 plate 3 8O<br />BBa C0060 plate 2 4H
| + | |
| − | </p>
| + | |
| − | <h1 class="day m40">6/7/15</h1>
| + | |
| − | <p class="content-txt">We transformed J61100, and spread it on the ampicillin medium.</p>
| + | |
| − | <h1 class="day m40">6/8/15</h1>
| + | |
| − | <p class="content-txt">We picked 3 colonies from J61100 and inoculated for the miniprep.</p>
| + | |
| − | <h1 class="day m40">6/11/15</h1>
| + | |
| − | <p class="content-txt">We inoculated J61100 for the midiprep.</p>
| + | |
| − | <h1 class="day m40">6/12/15</h1>
| + | |
| − | <p class="content-txt">We ran a midiprep on the J61100.</p>
| + | |
| − | <h1 class="day m40">6/15/15</h1>
| + | |
| − | <h2 class="sub-title">Enzyme digestion</h2>
| + | |
| − | <p class="content-txt">We digested J61100 with SpeI and PstI (used as a vector), while we digested C0062 with XbaI and PstI(used as a fragment), and digested C0060 (used as a fragment) with a XbaI and PstI. We then ran a gel electrophoresis and then extracted the gel for the plasmid fragment.</p>
| + | |
| − | <h2 class="sub-title">Ligation</h2>
| + | |
| − | <p class="content-txt">We ligated J61100 and C0062 while we ligated J61100 and C0060.</p>
| + | |
| − | <h2 class="sub-title">Transformation</h2>
| + | |
| − | <p class="content-txt">We then ran a transformation of these onto some competent cells.</p>
| + | |
| − | <h1 class="day m40">6/16/15</h1>
| + | |
| − | <p class="content-txt">We picked 6 colonies of each of J61100+C0062 and J61100+C0060, and inoculated them on the liquid LB. We then incubated the cell culture overnight for 16 hours.</p>
| + | |
| − | <h1 class="day m40">6/17/15</h1>
| + | |
| − | <p class="content-txt">We conducted a mini prep on J61100+C0062 and J61100+C0060.</p>
| + | |
| − | <h2 class="sub-title">Enzyme digestion</h2>
| + | |
| − | <p class="content-txt">We digested J37032 with XbaI and PstI (used as a fragment), and K823017 with SpeI and PstI (used as a vector). We then ran a gel electrophoresis and then extracted the gel for the plasmid fragment.</p>
| + | |
| − | <h2 class="sub-title">Ligation</h2>
| + | |
| − | <p class="content-txt">We ligated J37032 and K823017.</p>
| + | |
| − | <h2 class="sub-title">Transformation</h2>
| + | |
| − | <p class="content-txt">We then ran a transformation of these onto some competent cells.</p>
| + | |
| − | <h1 class="day m40">6/18/15</h1>
| + | |
| − | <p class="content-txt">We picked up 6 colonies of J37032+K823017 and inoculated them on the liquid LB. We then incubated the cell culture overnight for 16 hours.</p>
| + | |
| − | <h1 class="day m40">6/19/15</h1>
| + | |
| − | <p class="content-txt">We conducted a mini prep on J37032+K823017</p>
| + | |
| − | <h1 class="day m40">6/22/15</h1>
| + | |
| − | <p class="content-txt">We chose the plasmid which has plasmid that we want, and then we cut those plasmids with EcoRI and PstI. After that, we sent them to the sequencing company.</p>
| + | |
| − | <h1 class="day m40">6/25/15</h1>
| + | |
| − | <p class="content-txt">We learned that J61100 had ampicillin expression so we changed the vector to have chloramphenicol resistance.</p>
| + | |
| − | <h1 class="day m40">6/29/15</h1>
| + | |
| − | <h2 class="sub-title">Enzyme digestion</h2>
| + | |
| − | <p class="content-txt">We cut J61100+C0062 with SpeI and PstI (used as the vector), while we cut K823017+J37032 with XbaI and PstI (used as the fragment). We then ran a gel electrophoresis and then extracted the gel for the plasmid fragment.</p>
| + | |
| − | <h2 class="sub-title">Ligation</h2>
| + | |
| − | <p class="content-txt">We ligated J61100+C0062 and K823017+J37032, creating the reporter cell.</p>
| + | |
| − | <h2 class="sub-title">Transformation</h2>
| + | |
| − | <p class="content-txt">We then ran a transformation of these onto some competent cells.</p>
| + | |
| − | <h1 class="day m40">6/30/15</h1>
| + | |
| − | <p class="content-txt">We picked up 6 colonies of the reporter cell and inoculated them on the liquid LB. We then incubated the cell culture overnight for 16 hours.</p>
| + | |
| − | <h1 class="day m40">7/1/15</h1>
| + | |
| − | <p class="content-txt">We conducted a mini prep on the reporter cell.</p>
| + | |
| − | <h1 class="day m40">7/2/15</h1>
| + | |
| − | <p class="content-txt">We chose the plasmid which has plasmid that we want, and then we cut those plasmids with EcoRI and PstI. After that, we sent them to the sequencing company.</p>
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| − | </div>
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| − | </body>
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| − | </html>
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