Difference between revisions of "Team:Lanzhou/Notebook"

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{{Lanzhou}}
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{{Lanzhou/Header}}
 
<html>
 
<html>
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<script>
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$('.page__header h1').text('Notebook');
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</script>
  
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<main class="page__content notebook">
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<div class="column full_size">
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<section class="mdc-theme--dark">
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<h3>April</h3>
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<h1>Notebook</h1>
+
<ul class="mdc-list mdc-list--two-line">
−
<p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p>
+
<li class="mdc-list-item mdc-ripple-surface mdc-list-item--opened">
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+
<span class="mdc-list-item__text">
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</div>
+
Week 1
−
<div class="clear"></div>
+
<span class="mdc-list-item__text__secondary">Apr 1</span>
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+
</span>
−
 
+
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
−
<div class="column half_size">
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</li>
−
<h5>What should this page have?</h5>
+
<div style="display: inherit;" class="detail mdc-elevation--z1">
−
<ul>
+
• Establish research project in group meeting.
−
<li>Chronological notes of what your team is doing.</li>
+
</div>
−
<li> Brief descriptions of daily important events.</li>
+
<li class="mdc-list-item mdc-ripple-surface">
−
<li>Pictures of your progress. </li>
+
<span class="mdc-list-item__text">
−
<li>Mention who participated in what task.</li>
+
Week 2
−
</ul>
+
<span class="mdc-list-item__text__secondary">Apr 2 ~ Apr 8</span>
−
 
+
</span>
−
</div>
+
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
−
 
+
</li>
−
<div class="column half_size">
+
<div class="detail mdc-elevation--z1">
−
<h5>Inspiration</h5>
+
• Prepare for experiment and confirm members of the division of labor.
−
<p>You can see what others teams have done to organize their notes:</p>
+
<li>Antibiotic(Amp,Kana,Chl,Tet).</li>
−
 
+
<li>prepare 5M IPTG.</li>
−
<ul>  
+
<li>prepare double distilled water, centrifuge tube, PCR tube sterilization, of alcohol cotton.</li>
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+
<li>Preparation of competent state.</li>
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+
</div>
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+
<li class="mdc-list-item mdc-ripple-surface">
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+
<span class="mdc-list-item__text">
−
</ul>
+
Week 3
−
 
+
<span class="mdc-list-item__text__secondary">Apr 9 ~ Apr 15</span>
−
</div>
+
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• New members acquaint the laboratory,and do some basic experimental to prepare for the IGEM.
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Apr 16 ~ Apr 22</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Verify the plasmids and primer in group meeting.
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 5
 +
<span class="mdc-list-item__text__secondary">Apr 23 ~ Apr 29</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Gene synthesis
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>May</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Apr 30 ~ May 6</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Direct mutagenesis
 +
<li>Site-directed mutagenesis Lon1, Lon2.and run the gel to proving the verification of the mutation.</li>
 +
• Extract plasmids<br>
 +
• Digestion
 +
<li>Digested pUC19 (EcoRI, PstI), LacI and gel extraction.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">May 7 ~ May 13</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Site-directed mutagenesis Lon1, Lon2.
 +
<li>make bacterium Lon1, Lon2, pET28a (+), pUc19, pETDuet amplified.</li>
 +
• Transformation
 +
<li>Transform the plasmids into E.Coli DH5a.</li>
 +
• Culture
 +
<li>pick a single clone on the transformed plate and culture in fluid culture medium.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">May 14 ~ May 20</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Ligation
 +
<li>Use protocol of the Quick Connect Enzyme Kit to connect pUC19+GFP，pUC19+LacI.</li>
 +
• Transformation
 +
<li>Transform the mutation,connect product and L4440 plasmids.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">May 21 ~ May 27</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Plasmids extraction
 +
<li>Extract the plasmids of Gmix and P with plasmids Mini Kit,then run the gel electrophoresis (Figure 1).</li>
 +
Gel electrophoresis
 +
<li>run the gel electrophoresis of Ga(1~5) (Figure2).</li>
 +
<li>run the gel electrophoresis of Gb (1~5) (Figure3).</li>
 +
• Gel purification of Ga（Figure4）and Gb (Figure4).
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure1.png" alt="">
 +
<p><b>Figure 1.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure2.png" alt="">
 +
<p><b>Figure 2.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure3.png" alt="">
 +
<p><b>Figure 3.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure4.png" alt="">
 +
<p><b>Figure 4.</b></p>
 +
</figure>
 +
</div>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 5
 +
<span class="mdc-list-item__text__secondary">May 28 ~ Jun 3</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Digestion
 +
<li>digest Gain E/S,Gbin E/S.</li>
 +
• Ligation
 +
<li>Ligate Ga with pUC19,Gb with pUC19,Ga+Gb with pUC19.</li>
 +
• Transformation
 +
<li>Transform Ga+pUC19 into DH5a,Gb+pUC19 into DH5a,Ga+Gb+pUC19 into DH5a.</li>
 +
• Run gel
 +
<li>Extract pUC19 into 6 tubes.</li>
 +
<li>Gel purification of puc19 (Figure 6&7), analyse the failure of digestion.</li>`
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure6.png" alt="">
 +
<p><b>Figure 6.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure7.png" alt="">
 +
<p><b>Figure 7.</b></p>
 +
</figure>
 +
</div>
 +
• Ligation
 +
<li>Ligated GaE/P with Puc19 E/P, GbE/P with pUC19 E/P, GaE/S+GbX/P with pUC19 E/P.then Transfer ligated product into the plate,and Cultivate them overnight.</li>
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>June</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Jun 4 ~ Jun 10</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• PCR
 +
<li>PCR Ga, Gb, P, then gel purification (Figure 8&9).</li>
 +
<li>PCR protocol</li>
 +
<img src="image/notebook/table1.png" alt="">
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure8.png" alt="">
 +
<p><b>Figure 8.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure9.png" alt="">
 +
<p><b>Figure 9.</b></p>
 +
</figure>
 +
</div>
 +
• Plasmids extraction
 +
<li>Plasmids extraction of pUC19+Ga，pUC19+Gb.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">Jun 11 ~ Jun 17</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Repeat-ligation
 +
<li>Ligate GaE/P with Puc19 E/P,GbE/P with pUC19 E/P,GaE/S+GbX/P with pUC19 E/P.
 +
<!-- then Transfer ligated product into the plate,and Cultivate them overnight. </li> -->
 +
• Gel purification<br>
 +
• Repeat digestion
 +
<li>Repeat pUC19+Ga.E/S,pUC19+Gb.X/P. (Figure10)</li>
 +
• Digestion
 +
<li>Restriction digest P+EP6,ECR+EP10,ECR+EP6,SHT+EP6,SHT+EP8 (Figure11&12&13).</li>
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure10.png" alt="">
 +
<p><b>Figure 10.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure11.png" alt="">
 +
<p><b>Figure 11.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure12.png" alt="">
 +
<p><b>Figure 12.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure13.png" alt="">
 +
<p><b>Figure 13.</b></p>
 +
</figure>
 +
</div>
 +
• Repeat PCR
 +
<li>Repeat Gb PCR then gel purification</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">Jun 18 ~ Jun 24</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Transformation
 +
<li>Transform ECR+L4440 and SHT+L4440into DH5a.</li>
 +
• Repeat PCR
 +
<li>repeat Gb PCR and P digest (Figure14&15).</li>
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure14.png" alt="">
 +
<p><b>Figure 14.</b></p>
 +
</figure>
 +
<figure>
 +
<img src="image/notebook/figure15.png" alt="">
 +
<p><b>Figure 15.</b></p>
 +
</figure>
 +
</div>
 +
• Ligation
 +
<li>ligate ECR+pUC19 (Figure16)</li>
 +
<div class="figure">
 +
<figure>
 +
<img src="image/notebook/figure16.png" alt="">
 +
<p><b>Figure 16.</b></p>
 +
</figure>
 +
</div>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Jun 25 ~ Jul 1</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Culture
 +
<li>Culture bacterium in glycerol:MIX,GMIX, B1.</li>
 +
• plasmids extraction
 +
<li>MIX,GMIX, B1.</li>
 +
• Digestion
 +
<li>Digest MIX in P/E product and gel purification.</li>
 +
• Transformation
 +
<li>Transform the produce into puC19,then pick the positive.</li>
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>July</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Jul 2 ~ Jul 8</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• PCR
 +
<li>Do GMIX PCR </li>
 +
• Transformation
 +
<li>Transform PCR GMIX and into puC19.</li>
 +
<li>Tansform RcsA，RBS，pUC19 into BL21.</li>
 +
• Digestion
 +
<li>L4440，GbEXSP，GaEXSP and B1EXSP pick the positive and digest.</li>
 +
<li>GbEXSP，GaEXSP and B1EXSP.</li>
 +
• Plasmids extraction
 +
<li>GbEXSP，GaEXSP and B1EXSP extract plasmids.</li>
 +
<li>Kil，GFP，RBS，OmpT.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">Jul 9 ~ Jul 15</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Transformation:OmpT，pUC19.
 +
<li>Culture 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.</li>
 +
<li>PCR loop120</li>
 +
• plasmids extraction
 +
<li>17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.</li>
 +
• Digestion
 +
<li>Ga, Ga2, Gb, Gb2, B1, B2, SHT, SHT2, Ecr, Ecr2, RcsA, pUC19，pSB3K3, pSB4K5, Loop120, Lysis1, lysis2, Terminator.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">Jul 16 ~ Jul 22</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Ligation
 +
<li>Ga2+T——Ga2T</li>
 +
<li>Gb2+T——Gb2T</li>
 +
<li>B2+T——B2T</li>
 +
<li>SHT2+T——S2T</li>
 +
<li>Ecr2+T——E2T</li>
 +
<li>Loop120+pUC19——Loop120</li>
 +
<li>BBa_B0030+17P: 06 (0.47)——0.47x0.6</li>
 +
<li>BBa_B0064+17P: 06 (0.47)——0.47x0.35</li>
 +
<li>RcsA+T——RcsAT</li>
 +
<li>Lysis1+pUC19——lysis1</li>
 +
<li>Lysis2+pUC19——lysis2</li>
 +
• Plasmids extraction
 +
<li>Extract plasmids of pSB1T3，pSB1K3，pSB1C3，pSB1A3，SHT.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Jul 23 ~ Jul 29</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• PCR
 +
<li>Ga2T,Gb2T,B2T,S2T,E2T,Loop120,RcsAT,lysis1,lysis2.</li>
 +
<li>Prepare the transform cell of M bacterium,and all the plasmids need to transform would be gone and well.</li>
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>August</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Jul 30 ~ Aug 5</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Human practice
 +
<li>Confirm the form for human practice in group meeting.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">Aug 6 ~ Aug 12</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Human practice
 +
<li>Design a questionnaire of transgenosis in public.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">Aug 13 ~ Aug 19</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Human practice
 +
<li>Go to some schools advertise our work.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Aug 20 ~ Aug 26</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• We went to FAFU for CCIC.
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 5
 +
<span class="mdc-list-item__text__secondary">Aug 27 ~ Sep 2</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Gene synthesis
 +
<li>Synthesize the gene sequence act on arabidopsis thaliana.</li>
 +
• Vector construction
 +
<li>Construct 3hree vectors producing dsRNA (Ecr-L4440, ECRL, ECRN)</li>
 +
• Experiment on aphids.
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>September</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Sep 3 ~ Sep 9</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Transformation
 +
<li>Transform carrier(Ecr-L4440, ECRL, ECRN) into E.coli M-JLAC19 (Dicer deficit type).</li>
 +
• Induce expression<br>
 +
• Try to express dsRNA form trxzDP.
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">Sep 10 ~ Sep 16</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Induce expression
 +
<li>trxzDP:product dsRNA by T7 bi-directional promoter.</li>
 +
<li>trxzL: product hpRNA by normal loop construction.</li>
 +
<li>trxzN: product hpRNA by intron loop construction.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">Sep 17 ~ Sep 23</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Induce expression
 +
<li>Ecr-L4440:product dsRNA by T7 bi-directional promoter.</li>
 +
<li>ECRL:product hpRNA by normal loop construction.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Sep 24 ~ Sep 30</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Express of dsRNA and hpRNA.
 +
</div>
 +
</ul>
 +
<hr class="mdc-list-divider">
 +
<h3>October</h3>
 +
<ul class="mdc-list mdc-list--two-line">
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 1
 +
<span class="mdc-list-item__text__secondary">Oct 1 ~ Oct 7</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Culture arabidopsis thaliana (Col 0)<br>
 +
• Vector construction
 +
<li>Construct vectors of dsRNA which act on arabidopsis thaliana.</li>
 +
• Transformation
 +
<li>Transform carrier(trxzDP, trxzL, trxzN) into E.coli M-JLAC19 (Dicer deficit type).</li>
 +
• Induce expression
 +
<li>induce the M-JLAC19 express dsRNA.</li>
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 2
 +
<span class="mdc-list-item__text__secondary">Oct 8 ~ Oct 14</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Express dsRNA and hpRNA.<br>
 +
• Prepare for the macro experiments validation.
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 3
 +
<span class="mdc-list-item__text__secondary">Oct 15 ~ Oct 21</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Improve parts<br>
 +
• Modelling work
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 4
 +
<span class="mdc-list-item__text__secondary">Oct 22 ~ Oct 28</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Modelling work<br>
 +
• Presentation work
 +
</div>
 +
<li class="mdc-list-item mdc-ripple-surface">
 +
<span class="mdc-list-item__text">
 +
Week 5
 +
<span class="mdc-list-item__text__secondary">Oct 29 ~ Sep 31</span>
 +
</span>
 +
<i class="mdc-list-item__end-detail material-icons">keyboard_arrow_down</i>
 +
</li>
 +
<div class="detail mdc-elevation--z1">
 +
• Modelling work<br>
 +
• Wiki & design work<br>
 +
• Presentation work
 +
</div>
 +
</ul>
 +
  </section>
 +
</main>
 
</html>
 
</html>
 +
{{Lanzhou/Footer}}

Revision as of 15:46, 25 October 2017

Lanzhou

Lanzhou2017

April

  • Week 1 Apr 1 keyboard_arrow_down
    • • Establish research project in group meeting.
  • Week 2 Apr 2 ~ Apr 8 keyboard_arrow_down
    • • Prepare for experiment and confirm members of the division of labor.
  • Antibiotic(Amp,Kana,Chl,Tet).
  • prepare 5M IPTG.
  • prepare double distilled water, centrifuge tube, PCR tube sterilization, of alcohol cotton.
  • Preparation of competent state.
  • Week 3 Apr 9 ~ Apr 15 keyboard_arrow_down
    • • New members acquaint the laboratory,and do some basic experimental to prepare for the IGEM.
  • Week 4 Apr 16 ~ Apr 22 keyboard_arrow_down
    • • Verify the plasmids and primer in group meeting.
  • Week 5 Apr 23 ~ Apr 29 keyboard_arrow_down
    • • Gene synthesis

May

  • Week 1 Apr 30 ~ May 6 keyboard_arrow_down
    • • Direct mutagenesis
  • Site-directed mutagenesis Lon1, Lon2.and run the gel to proving the verification of the mutation.
    • • Extract plasmids
    • Digestion
  • Digested pUC19 (EcoRI, PstI), LacI and gel extraction.
  • Week 2 May 7 ~ May 13 keyboard_arrow_down
    • • Site-directed mutagenesis Lon1, Lon2.
  • make bacterium Lon1, Lon2, pET28a (+), pUc19, pETDuet amplified.
    • • Transformation
  • Transform the plasmids into E.Coli DH5a.
    • • Culture
  • pick a single clone on the transformed plate and culture in fluid culture medium.
  • Week 3 May 14 ~ May 20 keyboard_arrow_down
    • • Ligation
  • Use protocol of the Quick Connect Enzyme Kit to connect pUC19+GFP，pUC19+LacI.
    • • Transformation
  • Transform the mutation,connect product and L4440 plasmids.
  • Week 4 May 21 ~ May 27 keyboard_arrow_down
    • • Plasmids extraction
  • Extract the plasmids of Gmix and P with plasmids Mini Kit,then run the gel electrophoresis (Figure 1).
    • Gel electrophoresis
  • run the gel electrophoresis of Ga(1~5) (Figure2).
  • run the gel electrophoresis of Gb (1~5) (Figure3).
    • • Gel purification of Ga（Figure4）and Gb (Figure4).

    Figure 1.

    Figure 2.

    Figure 3.

    Figure 4.

  • Week 5 May 28 ~ Jun 3 keyboard_arrow_down
    • • Digestion
  • digest Gain E/S,Gbin E/S.
    • • Ligation
  • Ligate Ga with pUC19,Gb with pUC19,Ga+Gb with pUC19.
    • • Transformation
  • Transform Ga+pUC19 into DH5a,Gb+pUC19 into DH5a,Ga+Gb+pUC19 into DH5a.
    • • Run gel
  • Extract pUC19 into 6 tubes.
  • Gel purification of puc19 (Figure 6&7), analyse the failure of digestion.
    • `

    Figure 6.

    Figure 7.

    • Ligation
  • Ligated GaE/P with Puc19 E/P, GbE/P with pUC19 E/P, GaE/S+GbX/P with pUC19 E/P.then Transfer ligated product into the plate,and Cultivate them overnight.

June

  • Week 1 Jun 4 ~ Jun 10 keyboard_arrow_down
    • • PCR
  • PCR Ga, Gb, P, then gel purification (Figure 8&9).
  • PCR protocol

    • Figure 8.

    Figure 9.

    • Plasmids extraction
  • Plasmids extraction of pUC19+Ga，pUC19+Gb.
  • Week 2 Jun 11 ~ Jun 17 keyboard_arrow_down
    • • Repeat-ligation
  • Ligate GaE/P with Puc19 E/P,GbE/P with pUC19 E/P,GaE/S+GbX/P with pUC19 E/P. • Gel purification
    • Repeat digestion
  • Repeat pUC19+Ga.E/S,pUC19+Gb.X/P. (Figure10)
    • • Digestion
  • Restriction digest P+EP6,ECR+EP10,ECR+EP6,SHT+EP6,SHT+EP8 (Figure11&12&13).

    • Figure 10.

    Figure 11.

    Figure 12.

    Figure 13.

    • Repeat PCR
  • Repeat Gb PCR then gel purification
  • Week 3 Jun 18 ~ Jun 24 keyboard_arrow_down
    • • Transformation
  • Transform ECR+L4440 and SHT+L4440into DH5a.
    • • Repeat PCR
  • repeat Gb PCR and P digest (Figure14&15).

    • Figure 14.

    Figure 15.

    • Ligation
  • ligate ECR+pUC19 (Figure16)

    • Figure 16.

  • Week 4 Jun 25 ~ Jul 1 keyboard_arrow_down
    • • Culture
  • Culture bacterium in glycerol:MIX,GMIX, B1.
    • • plasmids extraction
  • MIX,GMIX, B1.
    • • Digestion
  • Digest MIX in P/E product and gel purification.
    • • Transformation
  • Transform the produce into puC19,then pick the positive.

July

  • Week 1 Jul 2 ~ Jul 8 keyboard_arrow_down
    • • PCR
  • Do GMIX PCR
    • • Transformation
  • Transform PCR GMIX and into puC19.
  • Tansform RcsA，RBS，pUC19 into BL21.
    • • Digestion
  • L4440，GbEXSP，GaEXSP and B1EXSP pick the positive and digest.
  • GbEXSP，GaEXSP and B1EXSP.
    • • Plasmids extraction
  • GbEXSP，GaEXSP and B1EXSP extract plasmids.
  • Kil，GFP，RBS，OmpT.
  • Week 2 Jul 9 ~ Jul 15 keyboard_arrow_down
    • • Transformation:OmpT，pUC19.
  • Culture 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
  • PCR loop120
    • • plasmids extraction
  • 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
    • • Digestion
  • Ga, Ga2, Gb, Gb2, B1, B2, SHT, SHT2, Ecr, Ecr2, RcsA, pUC19，pSB3K3, pSB4K5, Loop120, Lysis1, lysis2, Terminator.
  • Week 3 Jul 16 ~ Jul 22 keyboard_arrow_down
    • • Ligation
  • Ga2+T——Ga2T
  • Gb2+T——Gb2T
  • B2+T——B2T
  • SHT2+T——S2T
  • Ecr2+T——E2T
  • Loop120+pUC19——Loop120
  • BBa_B0030+17P: 06 (0.47)——0.47x0.6
  • BBa_B0064+17P: 06 (0.47)——0.47x0.35
  • RcsA+T——RcsAT
  • Lysis1+pUC19——lysis1
  • Lysis2+pUC19——lysis2
    • • Plasmids extraction
  • Extract plasmids of pSB1T3，pSB1K3，pSB1C3，pSB1A3，SHT.
  • Week 4 Jul 23 ~ Jul 29 keyboard_arrow_down
    • • PCR
  • Ga2T,Gb2T,B2T,S2T,E2T,Loop120,RcsAT,lysis1,lysis2.
  • Prepare the transform cell of M bacterium,and all the plasmids need to transform would be gone and well.

August

  • Week 1 Jul 30 ~ Aug 5 keyboard_arrow_down
    • • Human practice
  • Confirm the form for human practice in group meeting.
  • Week 2 Aug 6 ~ Aug 12 keyboard_arrow_down
    • • Human practice
  • Design a questionnaire of transgenosis in public.
  • Week 3 Aug 13 ~ Aug 19 keyboard_arrow_down
    • • Human practice
  • Go to some schools advertise our work.
  • Week 4 Aug 20 ~ Aug 26 keyboard_arrow_down
    • • We went to FAFU for CCIC.
  • Week 5 Aug 27 ~ Sep 2 keyboard_arrow_down
    • • Gene synthesis
  • Synthesize the gene sequence act on arabidopsis thaliana.
    • • Vector construction
  • Construct 3hree vectors producing dsRNA (Ecr-L4440, ECRL, ECRN)
    • • Experiment on aphids.

September

  • Week 1 Sep 3 ~ Sep 9 keyboard_arrow_down
    • • Transformation
  • Transform carrier(Ecr-L4440, ECRL, ECRN) into E.coli M-JLAC19 (Dicer deficit type).
    • • Induce expression
    • Try to express dsRNA form trxzDP.
  • Week 2 Sep 10 ~ Sep 16 keyboard_arrow_down
    • • Induce expression
  • trxzDP:product dsRNA by T7 bi-directional promoter.
  • trxzL: product hpRNA by normal loop construction.
  • trxzN: product hpRNA by intron loop construction.
  • Week 3 Sep 17 ~ Sep 23 keyboard_arrow_down
    • • Induce expression
  • Ecr-L4440:product dsRNA by T7 bi-directional promoter.
  • ECRL:product hpRNA by normal loop construction.
  • Week 4 Sep 24 ~ Sep 30 keyboard_arrow_down
    • • Express of dsRNA and hpRNA.

October

  • Week 1 Oct 1 ~ Oct 7 keyboard_arrow_down
    • • Culture arabidopsis thaliana (Col 0)
    • Vector construction
  • Construct vectors of dsRNA which act on arabidopsis thaliana.
    • • Transformation
  • Transform carrier(trxzDP, trxzL, trxzN) into E.coli M-JLAC19 (Dicer deficit type).
    • • Induce expression
  • induce the M-JLAC19 express dsRNA.
  • Week 2 Oct 8 ~ Oct 14 keyboard_arrow_down
    • • Express dsRNA and hpRNA.
    • Prepare for the macro experiments validation.
  • Week 3 Oct 15 ~ Oct 21 keyboard_arrow_down
    • • Improve parts
    • Modelling work
  • Week 4 Oct 22 ~ Oct 28 keyboard_arrow_down
    • • Modelling work
    • Presentation work
  • Week 5 Oct 29 ~ Sep 31 keyboard_arrow_down
    • • Modelling work
    • Wiki & design work
    • Presentation work