Team:KAIST Korea/Notebook Labnote/2012 8

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<section id="1">
<section id="1">
<div class="date">August 1<sup>st</sup> 2012</div></br>
<div class="date">August 1<sup>st</sup> 2012</div></br>
 +
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene : decreasing annealing temperature down to 45℃</div>
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 +
<div id="content_note" >
 +
 +
<span id="little">To make sure there’s no problem with unspecific binding or annealing temperature, we decreased annealing condition down to 45℃.</span>
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</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0801Fig1" src="http://2012.igem.org/wiki/images/e/e1/KAIST_08012012_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">There was no band and it became certain that there are no problems with annealing procedure.</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">Induction of <i>Moth_0109</i> gene (sampling) and running SDS-PAGE gel. </div>
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 +
<div id="content_note" >
 +
 +
<span id="little">We induced <i>Moth_0109</i> gene in pBAD/myc vector with different Arabinose conditions and temperature: 10mM, 30mM and 37℃, 30℃. And then, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.</span>
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</br></br>
 +
</div>
 +
</br><hr></br>
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 +
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene with Gibson and TOPO primer</div>
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 +
<div id="content_note" >
 +
 +
<span id="little">To see whether there are any problem with Gibson primers that we’ve designed, we did one more pcr amplification. Primers for Gibson assembly and TOPO cloning are mixed, and we could see any problems with forward or reverse primers. </span>
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</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0801Fig2" src="http://2012.igem.org/wiki/images/6/62/KAIST_08012012_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">Each lane indicates:</br></br>
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<ul style="list-style-type:square">
 +
<li><i>Moth_1191_1</i> : TOPO forward primer and Gibson reverse primer</li>
 +
<li><i>Moth_1191_2</i> : Gibson forward primer and TOPO reverse primer</li>
 +
<li><i>Moth_1199_1</i> : Gibson forward primer and TOPO reverse primer</li>
 +
<li><i>Moth_1199_2</i> : TOPO forward primer and Gibson reverse primer</li>
 +
</ul>
 +
</br>
 +
Correct size of band appeared when topo forward primer and Gibson reverse primer was used in amplification of both genes. Therefore, we can conclude that there is some unknown problem with forward primers.</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="2">
<section id="2">
<div class="date">August 2<sup>nd</sup> 2012</div></br>
<div class="date">August 2<sup>nd</sup> 2012</div></br>
 +
<div class="note-title">Primer design for FDH derived from <i>Candida boidinii</i> mutant</div>
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<div id="content_note" >
 +
<b>Results</b></br></br>
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 +
<div align="center"><img id="figure" alt="0802Fig1" src="http://2012.igem.org/wiki/images/b/b8/KAIST_08022012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">These primers are universial primer that can be used for insertion of this gene into both pTrcHis2A vector and pBAD/mycHisC vector. </span>
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 +
</br>
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 +
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</div>
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 +
</br><hr></br>
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 +
<div class="note-title">Expression check of <i>Moth_0109</i> gene on pBAD/mycHisC vector (Result)</div>
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 +
<div id="content_note" >
 +
 +
<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase. </span>
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</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0802Fig2" src="http://2012.igem.org/wiki/images/7/7c/KAIST_08022012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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 +
</br>
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">The band of PAGE gel was not certain, so we </span>
 +
<!--이것도 쓰다가 말았음;; -->
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</br>
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="3">
<section id="3">
<div class="date">August 3<sup>rd</sup> 2012</div></br>
<div class="date">August 3<sup>rd</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
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</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
 +
<section id="4">
<section id="4">
<div class="date">August 4<sup>th</sup> 2012</div></br>
<div class="date">August 4<sup>th</sup> 2012</div></br>
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<div class="note-title"><i>1191</i> and <i>1199</i> forward primer design</div>
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<div id="content_note" >
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 +
<span id="little">We designed new forward primers for both <i>1191</i> and <i>1199</i> gene that are thought to solve problems appeared during PCR amplification.</span>
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</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0804Fig1" src="http://2012.igem.org/wiki/images/5/59/KAIST_08042012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="5">
<section id="5">
<div class="date">August 5<sup>th</sup> 2012</div></br>
<div class="date">August 5<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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="6">
<section id="6">
<div class="date">August 6<sup>th</sup> 2012</div></br>
<div class="date">August 6<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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="7">
<section id="7">
<div class="date">August 7<sup>th</sup> 2012</div></br>
<div class="date">August 7<sup>th</sup> 2012</div></br>
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 +
<div class="note-title">FDH gene PCR amplification</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0807Fig1" src="http://2012.igem.org/wiki/images/7/7b/KAIST_08072012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">We amplified 2 samples of formate dehydrogenase gene with the primers we designed. From gel electrophoresis picture, we can say that oligonucleotides with the proper size 1100bp are present in the sample.</span>
 +
<div align="center"><img id="figure" alt="0807Fig2" src="http://2012.igem.org/wiki/images/e/eb/KAIST_08072012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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 +
</br>
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 +
<!--아 문법이상..졸려.왜 insert sample 이 하나밖에 없지. PCR purification 할 때 하나에 몰아서 했나?-->
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 +
</br>
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 +
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">August 8<sup>th</sup> 2012</div></br>
<div class="date">August 8<sup>th</sup> 2012</div></br>
 +
<div class="note-title">FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Procedure</b></br></br>
 +
 +
<span id="little">We mixed restriction enzymes and buffers with DNA samples as shown below.</span></br></br>
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 +
<div align="center"><img id="figure" alt="0808Fig1" src="http://2012.igem.org/wiki/images/3/31/KAIST_08082012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<span id="little">After mixing the enzymes and buffer with the sample, we incubated the sample on 37℃ for 1 hour and then on 65℃ for 20minutes. </span>
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 +
</br>
 +
 +
 +
</div>
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</br><hr></br>
 +
 +
<div class="note-title">pTrcHis2A, pBAD/myc vector dephosphorylation</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Procedure</b></br></br>
 +
<span id="little">We mixed phosphatidase and buffer with DNA sample as shown below.</span></br></br>
 +
<div align="center"><img id="figure" alt="0808Fig2" src="http://2012.igem.org/wiki/images/4/49/KAIST_08082012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">After mixing the enzyme and buffer with the sample, we incubated the sample on 37℃ for 1 hour 20minutes and then on 70℃ for 5minutes. </span>
 +
 +
</br>
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</div>
 +
</br><hr></br>
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 +
<div class="note-title">ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Procedure</b></br></br>
 +
<span id="little">We mixed ligase and buffer with DNA sample as shown below.</span>
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</br></br>
 +
<div align="center"><img id="figure" alt="0808Fig3" src="http://2012.igem.org/wiki/images/4/4e/KAIST_08082012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">After mixing the enzyme and buffer with the sample, we incubated the sample on 16℃ for 16 hours.</span>
 +
 +
</br>
 +
</div>
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 +
</br><hr></br>
 +
 +
<div class="note-title">Primer amplification of <i>1191</i> and <i>1199</i></div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0808Fig4" src="http://2012.igem.org/wiki/images/5/5d/KAIST_08082012_Fig4.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">These </span>
 +
<!--이것도 쓰다가 말았음-->
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
</section>
 +
<section id="9">
<section id="9">
<div class="date">August 9<sup>th</sup> 2012</div></br>
<div class="date">August 9<sup>th</sup> 2012</div></br>
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 +
<div class="note-title">vector transformation into TOP10 cell</div>
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 +
<div id="content_note" >
 +
 +
<span id="little">Transformation of pTrcHis2A vector into TOP10 cell was successful. So we did vector mini-preparation after the transformation. However, transformation of pBAD vector has failed, so we ligated the samples again. </span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0809Fig1" src="http://2012.igem.org/wiki/images/e/e9/KAIST_08092012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
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 +
</br>
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 +
<span id="little">This is gel picture to confirm that FDH is well inserted into TOP10 cells. 3 wells on the left are PCR product of <!--??/--></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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="10">
<section id="10">
<div class="date">August 10<sup>th</sup> 2012</div></br>
<div class="date">August 10<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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
</section>
<section id="11">
<section id="11">
<div class="date">August 11<sup>th</sup> 2012</div></br>
<div class="date">August 11<sup>th</sup> 2012</div></br>
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 +
<div class="note-title">pTrcHis2A vector transformation into <i>E.coli</i> MG1655 cell</div>
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 +
<div id="content_note" >
 +
<b>Procedure</b></br></br>
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">pBAD/mycHisC-FDH transformation into TOP10</div>
 +
 +
<div id="content_note" >
 +
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
 +
 +
<section id="12">
<section id="12">
<div class="date">August 12<sup>th</sup> 2012</div></br>
<div class="date">August 12<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">colony PCR check of pTrcHis2A inserted FDH gene</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0812Fig1" src="http://2012.igem.org/wiki/images/8/80/KAIST_08122012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115bp, which means formate dehydrogenase is inserted correctly. </span>
 +
 +
</br>
 +
 +
 +
</div>
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 +
</br><hr></br>
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 +
<div class="note-title">pBAD FDH MG1655 electroporation</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
 +
<span id="little">Fail</span>
 +
<!--여기 쓸말 더 있으면 써줘요 지환오빠 ㅋㅋ-->
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="13">
<section id="13">
<div class="date">August 13<sup>th</sup> 2012</div></br>
<div class="date">August 13<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">FDH pBAD MG1655 electroporation</div>
 +
 +
<div id="content_note" >
 +
<b>Procedure</b></br></br>
 +
 +
<span id="little">We seeded cells on 7:10 AM and did cell-down on 10:00AM. And then we plated cells on 1:00PM and checked colony on 8/14 5:00AM</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
<!--
 +
<div align="center"><img id="figure" alt="08##Fig#" src="#"></img></div>
 +
<div style="clear:both;"></div>    Colony pic.-->
 +
</br>
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">FDH pTrc MG1655 expression check (sampling and induction)</div>
 +
 +
<div id="content_note" >
 +
<b>Procedure</b></br></br>
 +
 +
<span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) on 7 : 30 AM and checked Optical Density on 10:30pm. According to the O.D. value, we induced 37℃ sample on 10:30PM and did sampling on 4:30pm. Also, we induced 30℃ sample on 11:30pm and did sampling on 5:30pm. (6 hour induction) Each temperature samples were induced by different conditions ; 2mM and 3mM of IPTG.</span>
 +
</br></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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">August 14<sup>th</sup> 2012</div></br>
<div class="date">August 14<sup>th</sup> 2012</div></br>
 +
<div class="note-title">colony PCR check of pBADmycHisC inserted FDH gene</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0814Fig1" src="http://2012.igem.org/wiki/images/e/e2/KAIST_08142012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115 bp, which means formate dehydrogenase is inserted correctly. </span>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0814Fig2" src="http://2012.igem.org/wiki/images/6/64/KAIST_08142012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 43.7 kDa protein, formate dehydrogenase. The band appeared significantly on the correct size. </span>
 +
 +
</br>
 +
</div>
 +
</br><hr></br>
 +
<div class="note-title">FDH pBAD MG1655 seeding for expression check</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Procedure</b></br></br>
 +
 +
<span id="little">We seeded cell on 7:00PM, so we’ll incubate cell about 16hours. (This means we have to do next seeding on 11:00AM. )</span>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">Primer design for the insertion of <i>1202</i> and <i>0109</i></div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0814Fig3" src="http://2012.igem.org/wiki/images/b/bd/KAIST_08142012_Fig3.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">To eliminate uncertain spacer between genes, we inserted </span>
 +
<!--쓰다가 말았네;;-->
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">August 15<sup>th</sup> 2012</div></br>
<div class="date">August 15<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">FDH pBAD MG1655 expression check</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Procedure</b></br></br>
 +
 +
<span id="little">We seeded cells on 11 : 30 AM and checked Optical Density on 1:30pm. According to the O.D. value, we induced 37℃ sample on 1:40pm and did sampling on 7:40pm. Also, we induced 30℃ sample on 3:30pm and did sampling on 9:30pm. (6 hour induction) Each temperature samples were induced by different conditions: 10mM of arabinose and 30mM of arabinose.</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">August 16<sup>th</sup> 2012</div></br>
<div class="date">August 16<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">PCR amplification of <i>1201</i>, <i>1202</i>, <i>1203</i>, <i>1198</i> and <i>1191</i></div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0816Fig1" src="http://2012.igem.org/wiki/images/8/86/KAIST_08152012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">Primers arrived, and we began to do additional PCR amplification on the preparation for Gibson assembly. Each PCR sample has the correct size of oligonucleotides except <i>Moth 1202</i> gene.</br></br>
 +
<ul>
 +
<li><i>1191</i> : 921bp</br></li>
 +
<li><i>1198</i> : 972bp + about 400bp = 1300 bp </br></li>
 +
<li><i>1201</i> : 1341bp + ?</br></li>
 +
<li><i>1202</i> : 2190bp + </br></li>
 +
<li><i>1203</i> : 2025bp + 1600bp = 3625bp</br></li>
 +
</ul>
 +
</br>
 +
We did PCR purification on the PCR product and the concentration/purity result was as shown below.
 +
</span>
 +
</br></br>
 +
 +
<div align="center"><img id="figure" alt="0816Fig2" src="http://2012.igem.org/wiki/images/3/30/KAIST_08162012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">Pre-culture of <i>0109</i></div>
 +
 +
<div id="content_note" >
 +
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">There was no vector prepared for Moth 0109, so we did 5ml pre-culture on 10ml tube. Cell was grown about 16hours after pre-culture, and we did vector mini-prep on the sample. The concentration of the mini-prepped vector is 726.8ng/ul and the purity(A<sub>260</sub>/A<sub>280</sub>) was 1.86.</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">August 17<sup>th</sup> 2012</div></br>
<div class="date">August 17<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">Expression check of Formate dehydrogenase.</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0817Fig1" src="http://2012.igem.org/wiki/images/3/34/KAIST_08172012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.xx kDa protein, formate dehydrogenase. The band appeared more significantly than the case of pTrcHis2A vector. Therefore, using pBAD promoter seems to produce more proteins.</span>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title">PCR amplification – second trial</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0817Fig2" src="http://2012.igem.org/wiki/images/5/5b/KAIST_08172012_Fig2.png" /></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">As the band was not significant for <i>1203</i> and <i>1198</i> on previous trial and purity was low for <i>1191</i>, we tried secondary PCR amplification on samples for Gibson assembly; <i>1202</i>, <i>1203</i>, <i>1198</i>, <i>1191</i>. Particularly for <i>1202</i> gene, we tried additional colony PCR, so that we could check any problem with vector mini-prep. (PCR solution for <i>1202</i> colony PCR was : 22ul </span>
 +
 +
</br></br>
 +
<div align="center"><img id="figure" alt="0817Fig3" src="http://2012.igem.org/wiki/images/e/e7/KAIST_08172012_Fig3.png" /></div>
 +
<div style="clear:both;"></div>
 +
 +
</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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="18">
<section id="18">
<div class="date">August 18<sup>th</sup> 2012</div></br>
<div class="date">August 18<sup>th</sup> 2012</div></br>
 +
 +
<div class="note-title">PCR amplification of <i>1202</i>, <i>0109</i> gene with original primer and Gibson primers</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0818Fig1" src="http://2012.igem.org/wiki/images/b/b0/KAIST_08182012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">Each lane indicates:</br></br>
 +
<ul>
 +
<li><i>Moth_1202_1</i> : Gibson forward + original reverse</li>
 +
<li><i>Moth_1202_2</i> : Gibson reverse + original forward</li>
 +
<li><i>Moth_0109_1</i> : Gibson forward + original reverse</li>
 +
<li><i>Moth_0109_2</i> : Gibson reverse + original forward</li>
 +
</ul>
 +
</br>
 +
There was correct size and unspecific size of band on <i>1202_1</i> sample. Although there are also unspecific band, <i>1202_2</i> sample has large band of correct size. <i>0109_1</i> sample has only unspecific band and <i>0109_2</i> sample has right size of band.
 +
</span>
 +
 +
</br></br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">When the PCR was unsuccessful, we first thought it might be problem of forward primer as in the case of <i>1199</i> and <i>1191</i>. Our guess was correct, and we’re going to fix the problem with forward primers. </span>
 +
 +
</div>
 +
 +
</br><hr></br>
 +
 +
<div class="note-title"><i>1202</i> and <i>0109</i> forward primer design</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">We designed new forward primer hoping no problem with it. </span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<!--여기부터 없음-->
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/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">
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<div class="date">August 31<sup>st</sup> 2012</div></br>
<div class="date">August 31<sup>st</sup> 2012</div></br>
</section>
</section>
 +
</br></br>
</br></br>
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<b>Results</b></br></br>
<b>Results</b></br></br>
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<div style="clear:both;"></div>
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<b>Results</b></br></br>
<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="08##Fig#" src="#"></img></div>
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<div style="clear:both;"></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="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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Revision as of 05:15, 15 September 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-August

Labnote

August

August 1st 2012

PCR amplification of Moth_1191, 1199 gene : decreasing annealing temperature down to 45℃
To make sure there’s no problem with unspecific binding or annealing temperature, we decreased annealing condition down to 45℃.

Results

0801Fig1

There was no band and it became certain that there are no problems with annealing procedure.



Induction of Moth_0109 gene (sampling) and running SDS-PAGE gel.
We induced Moth_0109 gene in pBAD/myc vector with different Arabinose conditions and temperature: 10mM, 30mM and 37℃, 30℃. And then, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.




PCR amplification of Moth_1191, 1199 gene with Gibson and TOPO primer
To see whether there are any problem with Gibson primers that we’ve designed, we did one more pcr amplification. Primers for Gibson assembly and TOPO cloning are mixed, and we could see any problems with forward or reverse primers.

Results

0801Fig2

Each lane indicates:

  • Moth_1191_1 : TOPO forward primer and Gibson reverse primer
  • Moth_1191_2 : Gibson forward primer and TOPO reverse primer
  • Moth_1199_1 : Gibson forward primer and TOPO reverse primer
  • Moth_1199_2 : TOPO forward primer and Gibson reverse primer

Correct size of band appeared when topo forward primer and Gibson reverse primer was used in amplification of both genes. Therefore, we can conclude that there is some unknown problem with forward primers.

Back to the Calendar
August 2nd 2012

Primer design for FDH derived from Candida boidinii mutant
Results

0802Fig1

Discussion

These primers are universial primer that can be used for insertion of this gene into both pTrcHis2A vector and pBAD/mycHisC vector.



Expression check of Moth_0109 gene on pBAD/mycHisC vector (Result)
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase.

Results

0802Fig2

Discussion

The band of PAGE gel was not certain, so we
Back to the Calendar
August 3rd 2012

No Special Event!
Back to the Calendar
August 4th 2012

1191 and 1199 forward primer design
We designed new forward primers for both 1191 and 1199 gene that are thought to solve problems appeared during PCR amplification.

Results

0804Fig1

Back to the Calendar
August 5th 2012

No Special Event!
Back to the Calendar
August 6th 2012

No Special Event!
Back to the Calendar
August 7th 2012

FDH gene PCR amplification
Results

0807Fig1

Discussion

We amplified 2 samples of formate dehydrogenase gene with the primers we designed. From gel electrophoresis picture, we can say that oligonucleotides with the proper size 1100bp are present in the sample.
0807Fig2


Back to the Calendar
August 8th 2012

FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion
Procedure

We mixed restriction enzymes and buffers with DNA samples as shown below.

0808Fig1

After mixing the enzymes and buffer with the sample, we incubated the sample on 37℃ for 1 hour and then on 65℃ for 20minutes.



pTrcHis2A, pBAD/myc vector dephosphorylation
Procedure

We mixed phosphatidase and buffer with DNA sample as shown below.

0808Fig2

After mixing the enzyme and buffer with the sample, we incubated the sample on 37℃ for 1 hour 20minutes and then on 70℃ for 5minutes.



ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors
Procedure

We mixed ligase and buffer with DNA sample as shown below.

0808Fig3

After mixing the enzyme and buffer with the sample, we incubated the sample on 16℃ for 16 hours.



Primer amplification of 1191 and 1199
Results

0808Fig4

Discussion

These
Back to the Calendar
August 9th 2012

vector transformation into TOP10 cell
Transformation of pTrcHis2A vector into TOP10 cell was successful. So we did vector mini-preparation after the transformation. However, transformation of pBAD vector has failed, so we ligated the samples again.

Results

0809Fig1

This is gel picture to confirm that FDH is well inserted into TOP10 cells. 3 wells on the left are PCR product of
Back to the Calendar
August 10th 2012

No Special Event!
Back to the Calendar
August 11th 2012

pTrcHis2A vector transformation into E.coli MG1655 cell
Procedure




pBAD/mycHisC-FDH transformation into TOP10

Back to the Calendar
August 12th 2012

colony PCR check of pTrcHis2A inserted FDH gene
Results

0812Fig1

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115bp, which means formate dehydrogenase is inserted correctly.



pBAD FDH MG1655 electroporation
Results

Fail
Back to the Calendar
August 13th 2012

FDH pBAD MG1655 electroporation
Procedure

We seeded cells on 7:10 AM and did cell-down on 10:00AM. And then we plated cells on 1:00PM and checked colony on 8/14 5:00AM

Results





FDH pTrc MG1655 expression check (sampling and induction)
Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) on 7 : 30 AM and checked Optical Density on 10:30pm. According to the O.D. value, we induced 37℃ sample on 10:30PM and did sampling on 4:30pm. Also, we induced 30℃ sample on 11:30pm and did sampling on 5:30pm. (6 hour induction) Each temperature samples were induced by different conditions ; 2mM and 3mM of IPTG.

Back to the Calendar
August 14th 2012

colony PCR check of pBADmycHisC inserted FDH gene
Results

0814Fig1

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115 bp, which means formate dehydrogenase is inserted correctly.



Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)
Results

0814Fig2

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 43.7 kDa protein, formate dehydrogenase. The band appeared significantly on the correct size.



FDH pBAD MG1655 seeding for expression check
Procedure

We seeded cell on 7:00PM, so we’ll incubate cell about 16hours. (This means we have to do next seeding on 11:00AM. )



Primer design for the insertion of 1202 and 0109
Results

0814Fig3

Discussion

To eliminate uncertain spacer between genes, we inserted
Back to the Calendar
August 15th 2012

FDH pBAD MG1655 expression check
Procedure

We seeded cells on 11 : 30 AM and checked Optical Density on 1:30pm. According to the O.D. value, we induced 37℃ sample on 1:40pm and did sampling on 7:40pm. Also, we induced 30℃ sample on 3:30pm and did sampling on 9:30pm. (6 hour induction) Each temperature samples were induced by different conditions: 10mM of arabinose and 30mM of arabinose.
Back to the Calendar
August 16th 2012

PCR amplification of 1201, 1202, 1203, 1198 and 1191
Results

0816Fig1

Discussion

Primers arrived, and we began to do additional PCR amplification on the preparation for Gibson assembly. Each PCR sample has the correct size of oligonucleotides except Moth 1202 gene.

  • 1191 : 921bp
  • 1198 : 972bp + about 400bp = 1300 bp
  • 1201 : 1341bp + ?
  • 1202 : 2190bp +
  • 1203 : 2025bp + 1600bp = 3625bp

We did PCR purification on the PCR product and the concentration/purity result was as shown below.


0816Fig2




Pre-culture of 0109
Discussion

There was no vector prepared for Moth 0109, so we did 5ml pre-culture on 10ml tube. Cell was grown about 16hours after pre-culture, and we did vector mini-prep on the sample. The concentration of the mini-prepped vector is 726.8ng/ul and the purity(A260/A280) was 1.86.
Back to the Calendar
August 17th 2012

Expression check of Formate dehydrogenase.
Results

0817Fig1

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.xx kDa protein, formate dehydrogenase. The band appeared more significantly than the case of pTrcHis2A vector. Therefore, using pBAD promoter seems to produce more proteins.



PCR amplification – second trial
Results

0817Fig2

Discussion

As the band was not significant for 1203 and 1198 on previous trial and purity was low for 1191, we tried secondary PCR amplification on samples for Gibson assembly; 1202, 1203, 1198, 1191. Particularly for 1202 gene, we tried additional colony PCR, so that we could check any problem with vector mini-prep. (PCR solution for 1202 colony PCR was : 22ul

0817Fig3

Back to the Calendar
August 18th 2012

PCR amplification of 1202, 0109 gene with original primer and Gibson primers
Results

0818Fig1

Each lane indicates:

  • Moth_1202_1 : Gibson forward + original reverse
  • Moth_1202_2 : Gibson reverse + original forward
  • Moth_0109_1 : Gibson forward + original reverse
  • Moth_0109_2 : Gibson reverse + original forward

There was correct size and unspecific size of band on 1202_1 sample. Although there are also unspecific band, 1202_2 sample has large band of correct size. 0109_1 sample has only unspecific band and 0109_2 sample has right size of band.


Discussion

When the PCR was unsuccessful, we first thought it might be problem of forward primer as in the case of 1199 and 1191. Our guess was correct, and we’re going to fix the problem with forward primers.



1202 and 0109 forward primer design
We designed new forward primer hoping no problem with it.

Results

Back to the Calendar
August 19th 2012

August 20th 2012

August 21st 2012

August 22nd 2012

August 23rd 2012

August 24th 2012

August 25th 2012

August 26th 2012

August 27th 2012

August 28th 2012

August 29th 2012

August 30th 2012

August 31st 2012



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