Team:KAIST Korea/Notebook Labnote/2012 6

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<section id="1">
<section id="1">
<div class="date">June 1<sup>st</sup> 2012</div></br>
<div class="date">June 1<sup>st</sup> 2012</div></br>
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<div class="note-title">Vector mini-prep: <i>Moth_gene</i> carrying pTrcHis2A transformed into TOP10 strain</div>
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<div id="content_note" >
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<span id="little">We extracted the vector carrying <i>Moth_1201</i>, <i>1202</i>, <i>1204</i>, <i>2314</i>, <i>1197</i>, <i>1198</i>, <i>1199</i>, <i>0109</i>, <i>1191</i>, <i>1516</i> from transformed <i>E. coli</i> TOP10 strain.</span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0601Fig1" src="https://static.igem.org/mediawiki/2012/c/c1/KAIST_06012012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title">PCR for TOPO cloning </div>
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<div id="content_note" >
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<span id="little">With the extracted vectors, we performed PCR to amplify each inserted genes to further use them for TOPO vector cloning. As we always do, elongation time was set as 1 min/kb. </span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0601Fig2" src="https://static.igem.org/mediawiki/2012/e/ed/KAIST_06012012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">With these amplified genes, we performed PCR purification. Concentration and purity are as follows.</span>
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</br></br>
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<div align="center"><img id="figure" alt="0601Fig3" src="https://static.igem.org/mediawiki/2012/e/ee/KAIST_06012012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="2">
<section id="2">
<div class="date">June 2<sup>nd</sup> 2012</div></br>
<div class="date">June 2<sup>nd</sup> 2012</div></br>
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<div class="note-title">PCR amplification of control gene</div>
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<div id="content_note" >
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<span id="little">We PCR amplified a gene to use as a negative control when evaluating the expression result of TOPO cloning.</span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0602Fig1" src="https://static.igem.org/mediawiki/2012/4/47/KAIST_06022012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">Control gene is about 800nt long. We purified this gene and its concentration was 216.0 ng/µl with purity of 1.80.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title">TOPO cloning </div>
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<div id="content_note" >
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<span id="little">With the PCR amplified and purified genes, we performed TOPO cloning by inserting those genes into pBAD TOPO vector to examine the expression of genes with different inducing agent. Ligated TOPO vector was transformed into <i>E. coli</i> TOP10 strain by heat shock transformation method. Transformed cells were spread on LB agar plate for further colony check.</span>
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</br>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_0109</i>, <i>1191</i>, <i>1516</i> expression check</div>
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<div id="content_note" >
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<span id="little">Cells transformed with pTrcHis2A vectors carrying each of the genes <i>Moth_0109</i>, <i>1191</i>, <i>1516</i> were seeded to examine the expression of each gene. As this vector carries lac operon, genes are induced by treating IPTG of proper amount. Six hours after induction, cell culture will be harvested and the amount of target proteins will be assessed by SDS PAGE. (Both soluble and insoluble fractions of proteins were assessed.)</span>
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</br></br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="3">
<section id="3">
<div class="date">June 3<sup>rd</sup> 2012</div></br>
<div class="date">June 3<sup>rd</sup> 2012</div></br>
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<div class="note-title">Colony PCR: 12 transformed genes and control gene</div>
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<div id="content_note" >
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<span id="little">We performed colony PCR with 12 transformed samples and a control to verify if the transformation of cloned TOPO vector into TOP10 strain was successful. Total three colonies were selected from each samples.</span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0603Fig1" src="https://static.igem.org/mediawiki/2012/d/df/KAIST_06032012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<div align="center"><img id="figure" alt="0603Fig2" src="https://static.igem.org/mediawiki/2012/a/a1/KAIST_06032012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<div align="center"><img id="figure" alt="0603Fig3" src="https://static.igem.org/mediawiki/2012/a/ab/KAIST_06032012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">Some bands of samples failed to appear in the right place such as <i>Moth_0109</i> and <i>2314</i>.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title">Re-colony PCR: <i>Moth_0109</i>, <i>2314</i></div>
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<div id="content_note" >
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<span id="little">Since two samples did not show any successful result in colony PCR and we just gave it another try. </span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0604Fig4" src="https://static.igem.org/mediawiki/2012/9/9f/KAIST_06032012_Fig4.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">As we can see, <i>Moth_0109</i> genes amplified through colony PCR has different length compared to the gene construct originally inserted into the vector. This means that <i>Moth_0109</i> failed to be cloned into the vector and thus transformation was not successful either.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_0109</i>, <i>1191</i>, <i>1516</i> SDS PAGE</div>
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<div id="content_note" >
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<span id="little">With the seeded samples on 6/2, we induced the cell cultures and performed SDS PAGE to see the expression of each gene.</span>
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</br></br>
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</div>
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</br><hr></br>
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<div class="note-title">Colony selection and seeding for sequencing</div>
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<div id="content_note" >
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<span id="little">From successfully transformed samples, we selected colonies and cultured them for sequencing. Once their vectors are extracted, they will be sent to Xenotech for sequencing. </span>
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</br></br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="4">
<section id="4">
<div class="date">June 4<sup>th</sup> 2012</div></br>
<div class="date">June 4<sup>th</sup> 2012</div></br>
 +
<div class="note-title">Colony PCR: <i>Moth_0109</i> (10 samples)</div>
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 +
<div id="content_note" >
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 +
<span id="little">Since <i>Moth_0109</i> gene did not show any satisfactory result from colony PCR, we tried it again to figure out the problem.</span>
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</br></br>
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<b>Results</b></br></br>
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 +
<div align="center"><img id="figure" alt="0604Fig1" src="https://static.igem.org/mediawiki/2012/6/61/KAIST_06042012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">PCR results showed multiple bands again. Moreover, bands are not in the correct position.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title">Vector mini-prep for sequencing</div>
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 +
<div id="content_note" >
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<span id="little">We extracted vectors from 11 successfully transformed samples of TOP10.</span>
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</br></br>
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<b>Results</b></br></br>
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 +
<div align="center"><img id="figure" alt="0604Fig2" src="https://static.igem.org/mediawiki/2012/6/67/KAIST_06042012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<div align="center"><img id="figure" alt="0604Fig3" src="https://static.igem.org/mediawiki/2012/b/be/KAIST_06042012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">Among these samples, those whose concentration exceeded 100 ng/µl were sent for sequencing.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="5">
<section id="5">
<div class="date">June 5<sup>th</sup> 2012</div></br>
<div class="date">June 5<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="6">
<section id="6">
<div class="date">June 6<sup>th</sup> 2012</div></br>
<div class="date">June 6<sup>th</sup> 2012</div></br>
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<div id="content_note" >
 +
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="7">
<section id="7">
<div class="date">June 7<sup>th</sup> 2012</div></br>
<div class="date">June 7<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="8">
<section id="8">
<div class="date">June 8<sup>th</sup> 2012</div></br>
<div class="date">June 8<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="9">
<section id="9">
<div class="date">June 9<sup>th</sup> 2012</div></br>
<div class="date">June 9<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="10">
<section id="10">
<div class="date">June 10<sup>th</sup> 2012</div></br>
<div class="date">June 10<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="11">
<section id="11">
<div class="date">June 11<sup>th</sup> 2012</div></br>
<div class="date">June 11<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="12">
<section id="12">
<div class="date">June 12<sup>th</sup> 2012</div></br>
<div class="date">June 12<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="13">
<section id="13">
<div class="date">June 13<sup>th</sup> 2012</div></br>
<div class="date">June 13<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="14">
<section id="14">
<div class="date">June 14<sup>th</sup> 2012</div></br>
<div class="date">June 14<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="15">
<section id="15">
<div class="date">June 15<sup>th</sup> 2012</div></br>
<div class="date">June 15<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="16">
<section id="16">
<div class="date">June 16<sup>th</sup> 2012</div></br>
<div class="date">June 16<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="17">
<section id="17">
<div class="date">June 17<sup>th</sup> 2012</div></br>
<div class="date">June 17<sup>th</sup> 2012</div></br>
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<div id="content_note" >
 +
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="18">
<section id="18">
<div class="date">June 18<sup>th</sup> 2012</div></br>
<div class="date">June 18<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No special event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="19">
<section id="19">
<div class="date">June 19<sup>th</sup> 2012</div></br>
<div class="date">June 19<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
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<span id="little">No special event!</span>
 +
</br>
 +
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="20">
<section id="20">
<div class="date">June 20<sup>th</sup> 2012</div></br>
<div class="date">June 20<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 +
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="21">
<section id="21">
<div class="date">June 21<sup>st</sup> 2012</div></br>
<div class="date">June 21<sup>st</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 +
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
 +
<section id="22">
<section id="22">
<div class="date">June 22<sup>nd</sup> 2012</div></br>
<div class="date">June 22<sup>nd</sup> 2012</div></br>
 +
 +
<div class="note-title"><i>Moth_0109</i>, <i>1202</i> colony PCR</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0622Fig1" src="https://static.igem.org/mediawiki/2012/b/b9/KAIST_06222012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">There is no band in the gel. We failed TOPO cloning of <i>Moth 0109</i>, <i>1202</i>.</span>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
 +
<section id="23">
<section id="23">
<div class="date">June 23<sup>rd</sup> 2012</div></br>
<div class="date">June 23<sup>rd</sup> 2012</div></br>
-
</section>
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-
<section id="24">
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<div class="note-title"><i>Moth_0109</i>, <i>1202</i> colony PCR</div>
-
<div class="date">June 24<sup>th</sup> 2012</div></br>
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-
</section>
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-
<section id="25">
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<div class="date">June 25<sup>th</sup> 2012</div></br>
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-
</section>
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<section id="26">
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<div class="date">June 26<sup>th</sup> 2012</div></br>
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</section>
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<section id="27">
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-
<div class="date">June 27<sup>th</sup> 2012</div></br>
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-
</section>
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<section id="28">
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-
<div class="date">June 28<sup>th</sup> 2012</div></br>
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-
</section>
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-
<section id="29">
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-
<div class="date">June 29<sup>th</sup> 2012</div></br>
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-
</section>
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<section id="30">
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<div class="date">June 30<sup>th</sup> 2012</div></br>
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</section>
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<section id="31">
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<div class="date">June 31<sup>st</sup> 2012</div></br>
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-
</section>
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</br></br>
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<div id="content_note" >
-
</div>
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-
</div>
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<b>Results</b></br></br>
-
</body>
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<div align="center"><img id="figure" alt="0623Fig1" src="https://static.igem.org/mediawiki/2012/3/3f/KAIST_06232012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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{{:Team:KAIST_Korea/footer}}
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<!--
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</br>
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##########SAMPLE!!!##########
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<div class="note-title">TITLE</div>
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<span id="little">There is no band in the gel. We failed TOPO cloning of <i>Moth 0109</i>, <i>1202</i>.</span>
 +
 +
</br>
 +
 
 +
 
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 
 +
</section>
 +
 
 +
<section id="24">
 +
<div class="date">June 24<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title"><i>Moth_0109</i>, <i>1202</i> PCR result</div>
<div id="content_note" >
<div id="content_note" >
-
<span id="little">CONTENT</span>
 
-
</br></br>
 
<b>Results</b></br></br>
<b>Results</b></br></br>
-
<div align="center"><img id="figure" alt="04##Fig#" src="#"></img></div>
+
<div align="center"><img id="figure" alt="0624Fig1" src="https://static.igem.org/mediawiki/2012/6/67/KAIST_06242012_Fig1.png"></img></div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
</br>
</br>
-
<span id="little"></span>
+
<span id="little">We failed <i>Moth_0109</i>, <i>1202</i> TOPO cloning so we tried it again. The PCR band is correct. It means we were successful to make insert DNA.</span>
</br>
</br>
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</br><hr></br>
</br><hr></br>
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<div class="note-title">TITLE2</div>
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<div class="note-title">pBAD TOPO enzyme cut extraction</div>
<div id="content_note" >
<div id="content_note" >
-
 
-
<span id="little">CONTENT2</span>
 
-
</br></br>
 
<b>Results</b></br></br>
<b>Results</b></br></br>
-
<div align="center"><img id="figure" alt="04##Fig#" src="#"></img></div>
+
<div align="center"><img id="figure" alt="0624Fig2" src="https://static.igem.org/mediawiki/2012/0/04/KAIST_06242012_Fig2.png"></img></div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
</br>
</br>
-
<span id="little"></span>
+
<span id="little">We didn’t have the TOPO solution anymore. So we tried to reuse the TOPO vector from the other cloned vector. We cut the vector using same restriction enzyme with <i>Moth 0109</i>, <i>1202</i> insert and extracted the vector.</span>
</br>
</br>
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<section id="25">
 +
<div class="date">June 25<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
 +
<section id="26">
 +
<div class="date">June 26<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
 +
<section id="27">
 +
<div class="date">June 27<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
 +
<section id="28">
 +
<div class="date">June 28<sup>th</sup> 2012</div></br>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
 +
<section id="29">
 +
<div class="date">June 29<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
 +
<section id="30">
 +
<div class="date">June 30<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
 +
<span id="little">No special event!</span>
 +
</br>
 +
 
 +
 
 +
</div>
 +
 
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
 +
</br></br>
 +
</div>
 +
</div>
 +
 
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</body>
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</html>
 +
{{:Team:KAIST_Korea/footer}}

Latest revision as of 12:24, 26 September 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-June

Labnote

June

June 1st 2012

Vector mini-prep: Moth_gene carrying pTrcHis2A transformed into TOP10 strain
We extracted the vector carrying Moth_1201, 1202, 1204, 2314, 1197, 1198, 1199, 0109, 1191, 1516 from transformed E. coli TOP10 strain.

Results

0601Fig1



PCR for TOPO cloning
With the extracted vectors, we performed PCR to amplify each inserted genes to further use them for TOPO vector cloning. As we always do, elongation time was set as 1 min/kb.

Results

0601Fig2

With these amplified genes, we performed PCR purification. Concentration and purity are as follows.

0601Fig3

Back to the Calendar
June 2nd 2012

PCR amplification of control gene
We PCR amplified a gene to use as a negative control when evaluating the expression result of TOPO cloning.

Results

0602Fig1

Control gene is about 800nt long. We purified this gene and its concentration was 216.0 ng/µl with purity of 1.80.


TOPO cloning
With the PCR amplified and purified genes, we performed TOPO cloning by inserting those genes into pBAD TOPO vector to examine the expression of genes with different inducing agent. Ligated TOPO vector was transformed into E. coli TOP10 strain by heat shock transformation method. Transformed cells were spread on LB agar plate for further colony check.



Moth_0109, 1191, 1516 expression check
Cells transformed with pTrcHis2A vectors carrying each of the genes Moth_0109, 1191, 1516 were seeded to examine the expression of each gene. As this vector carries lac operon, genes are induced by treating IPTG of proper amount. Six hours after induction, cell culture will be harvested and the amount of target proteins will be assessed by SDS PAGE. (Both soluble and insoluble fractions of proteins were assessed.)

Back to the Calendar
June 3rd 2012

Colony PCR: 12 transformed genes and control gene
We performed colony PCR with 12 transformed samples and a control to verify if the transformation of cloned TOPO vector into TOP10 strain was successful. Total three colonies were selected from each samples.

Results

0603Fig1

0603Fig2

0603Fig3

Some bands of samples failed to appear in the right place such as Moth_0109 and 2314.


Re-colony PCR: Moth_0109, 2314
Since two samples did not show any successful result in colony PCR and we just gave it another try.

Results

0604Fig4

As we can see, Moth_0109 genes amplified through colony PCR has different length compared to the gene construct originally inserted into the vector. This means that Moth_0109 failed to be cloned into the vector and thus transformation was not successful either.


Moth_0109, 1191, 1516 SDS PAGE
With the seeded samples on 6/2, we induced the cell cultures and performed SDS PAGE to see the expression of each gene.



Colony selection and seeding for sequencing
From successfully transformed samples, we selected colonies and cultured them for sequencing. Once their vectors are extracted, they will be sent to Xenotech for sequencing.

Back to the Calendar
June 4th 2012

Colony PCR: Moth_0109 (10 samples)
Since Moth_0109 gene did not show any satisfactory result from colony PCR, we tried it again to figure out the problem.

Results

0604Fig1

PCR results showed multiple bands again. Moreover, bands are not in the correct position.


Vector mini-prep for sequencing
We extracted vectors from 11 successfully transformed samples of TOP10.

Results

0604Fig2

0604Fig3

Among these samples, those whose concentration exceeded 100 ng/µl were sent for sequencing.
Back to the Calendar
June 5th 2012

No special event!
Back to the Calendar
June 6th 2012

No special event!
Back to the Calendar
June 7th 2012

No special event!
Back to the Calendar
June 8th 2012

No special event!
Back to the Calendar
June 9th 2012

No special event!
Back to the Calendar
June 10th 2012

No special event!
Back to the Calendar
June 11th 2012

No special event!
Back to the Calendar
June 12th 2012

No special event!
Back to the Calendar
June 13th 2012

No special event!
Back to the Calendar
June 14th 2012

No special event!
Back to the Calendar
June 15th 2012

No special event!
Back to the Calendar
June 16th 2012

No special event!
Back to the Calendar
June 17th 2012

No special event!
Back to the Calendar
June 18th 2012

No special event!
Back to the Calendar
June 19th 2012

No special event!
Back to the Calendar
June 20th 2012

No special event!
Back to the Calendar
June 21st 2012

No special event!
Back to the Calendar
June 22nd 2012

Moth_0109, 1202 colony PCR
Results

0622Fig1

There is no band in the gel. We failed TOPO cloning of Moth 0109, 1202.
Back to the Calendar
June 23rd 2012

Moth_0109, 1202 colony PCR
Results

0623Fig1

There is no band in the gel. We failed TOPO cloning of Moth 0109, 1202.
Back to the Calendar
June 24th 2012

Moth_0109, 1202 PCR result
Results

0624Fig1

We failed Moth_0109, 1202 TOPO cloning so we tried it again. The PCR band is correct. It means we were successful to make insert DNA.


pBAD TOPO enzyme cut extraction
Results

0624Fig2

We didn’t have the TOPO solution anymore. So we tried to reuse the TOPO vector from the other cloned vector. We cut the vector using same restriction enzyme with Moth 0109, 1202 insert and extracted the vector.
Back to the Calendar
June 25th 2012

No special event!
Back to the Calendar
June 26th 2012

No special event!
Back to the Calendar
June 27th 2012

No special event!
Back to the Calendar
June 28th 2012

No special event!
Back to the Calendar
June 29th 2012

No special event!
Back to the Calendar
June 30th 2012

No special event!
Back to the Calendar


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