Team:ZJU-China/notebook.htm

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               <h2 class="acc_trigger"><a href="http://thesum.ca/work.html#">01 <strong>BRAINSTORM</strong></a></h2>
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               <h2 class="acc_trigger">01 <strong>BRAINSTORM</strong></h2>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm1.htm">Controlled chemistry plant</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm1.htm" style="text-decoration:none">Controlled chemistry plant</a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm2.htm">Synchronized bacterial clock</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm2.htm" style="text-decoration:none">Synchronized bacterial clock</a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm3.htm">EPad</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm3.htm" style="text-decoration:none">EPad</a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm4.htm">Methane alarm</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm4.htm" style="text-decoration:none">Methane alarm</a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm5.htm">Silkworm</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm5.htm" style="text-decoration:none">Silkworm</a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm6.htm">Radio-communication </a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm6.htm" style="text-decoration:none">Radio-communication </a>
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<a target="idFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm7.htm">Plant doctor</a>
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<a target="brainFrame" href="http://2012.igem.org/Team:ZJU-China/brainstorm7.htm" style="text-decoration:none">Plant doctor</a>
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           <h2 class="acc_trigger"><a href="http://thesum.ca/work.html#">02 <strong>PROTOCAL</strong></a></h2>
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           <h2 class="acc_trigger">02 <strong>PROTOCAL</strong></h2>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_1.htm" style="text-decoration:none">1 Stock solutions</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_2.htm" style="text-decoration:none">2 Antibiotics</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_3.htm" style="text-decoration:none">3 Cell transformation</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_4.htm" style="text-decoration:none">4 Glycerol stock</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_5.htm" style="text-decoration:none">5 Miniprep</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_6.htm" style="text-decoration:none">6 DNA Agarose Gels</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_7.htm" style="text-decoration:none">7 PCR</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_8.htm" style="text-decoration:none">8 PCR Purification</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_9.htm" style="text-decoration:none">9 Restriction Digest</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_10.htm" style="text-decoration:none">10 Gel Extraction</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_11.htm" style="text-decoration:none">11 Ligation</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_12.htm" style="text-decoration:none">12 Growth Curve</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_13.htm" style="text-decoration:none">13 Split GFP experiments</a>
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           <h2 class="acc_trigger"><a href="http://thesum.ca/work.html#">03 <strong>LAB NOTE</strong></a></h2>
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           <h2 class="acc_trigger">03 <strong>LAB NOTE</strong></h2>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote1.htm" style="text-decoration:none">Week 1</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote2.htm" style="text-decoration:none">Week 2</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote3.htm" style="text-decoration:none">Week 3</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote4.htm" style="text-decoration:none">Week 4</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote5.htm" style="text-decoration:none">Week 5</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote6.htm" style="text-decoration:none">Week 6</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote7.htm" style="text-decoration:none">Week 7</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote8.htm" style="text-decoration:none">Week 8</a>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote9.htm" style="text-decoration:none">Week 9</a>
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<h2>Week 1 (06.22-06.24): Preparation</h2>
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<h3>June 22</h3>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote10.htm" style="text-decoration:none">Week 10</a>
-
<p>1. Do some washing.</p>
+
<br>
-
<p>2. Prepare LB plates, including ampicillin, kanamycin and spectinomycin resistance.</p>
+
<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote11.htm" style="text-decoration:none">Week 11</a>
-
<p>3. Get some E.coli DH5&alpha;competent cells from Prof. Xiao Mu. Receive E.coli BL21*(DE3) strain requested previously.</p>
+
<br>
-
<p>4. Transformation: extract plasmid pCJDD0 (Ampr), pCJDFA (Kanr) and pCJDFB (Spr) from filter paper received, transform them into DH5&alpha;competent cells respectively.</p>
+
<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote12.htm" style="text-decoration:none">Week 12</a>
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<br>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote13.htm" style="text-decoration:none">Week 13</a>
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<br>
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<a target="labFrame" href="http://2012.igem.org/Team:ZJU-China/labnote14.htm" style="text-decoration:none">Week 14</a>
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<h3>June 23</h3>
 
-
<p>Amplify E.coli BL21*(DE3) strain.</p>
 
-
<h3>June 24</h3>
 
-
<p>1. Make glycerol stock of E.coli BL21*(DE3) strain.</p>
 
-
<p>2. Make competent cells of E.coli BL21*(DE3) strain and DH10&beta;.</p>
 
-
<h2>Week 2 (06.25-07.01): Amplify pCJDD0, pCJDFA and pCJDFB</h2>
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<h3>June 25</h3>
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<p>Pick up single colony of pCJDD0 and pCJDFA from plate of June 22 and amplify them in liquid LB medium.</p>
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<h3>June 26</h3>
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<iframe src="" frameborder="0" name="labFrame" width="100%" height="1000px"> </iframe>  
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<p>1. Transform pCJDD0 (Ampr), pCJDFA (Spr) and pCJDFB (Kanr) into DH5&alpha; and DH10&beta; respectively and adjust the resistance from that of June 22 because of a small mistake in supplementary information of reference.</p>
+
-
<p>2. Miniprep plasmid of pCJDD0 from bacteria of June25.</p>
+
-
<p>Name: D0-6-26-1      Concentration: 27.5 ng/ul      A260/280: 1.76</p>
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-
<p>Name: D0-6-26-2      Concentration: 24.7 ng/ul      A260/280: 1.81</p>
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-
<p>3. Transform plasmid D0-6-26-2 into DH10&beta;</p>
+
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<h3>June 27</h3>
+
-
<p>1. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH10&beta;), pCJDFB (DH10&beta;) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.</p>
+
-
<p>2. Miniprep plasmid of D0-6-26-2 from bacteria of June27.</p>
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-
<p>Name: D0-6-27-1      Concentration: 3.0 ng/ul      A260/280: 2.36</p>
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-
<p>Name: D0-6-27-2      Concentration: 2.0 ng/ul      A260/280: -8.61</p>
+
-
<p>Name: D0-6-27-3      Concentration: 3.6 ng/ul      A260/280: 1.70</p>
+
-
<p>Name: D0-6-27-4      Concentration: 3.8 ng/ul      A260/280: 1.73</p>
+
-
<p>We forget to add ethanol in Solution W2 of Axygen Miniprep Kit, leading the failure of the experiment.</p>
+
-
<h3>June 28</h3>
+
-
<p>1. Miniprep plasmid of D0-6-26-2 and pCJDFA from bacteria of June27.</p>
+
-
<p>Name: D0-6-28-1      Concentration: 16.2 ng/ul      A260/280: 2.11</p>
+
-
<p>Name: D0-6-28-2      Concentration: 21.0 ng/ul      A260/280: 1.80</p>
+
-
<p>[Sequencing]</p>
+
-
<p>Name: D0-6-28-3      Concentration: 21.2 ng/ul      A260/280: 1.77 </p>
+
-
<p>[Sequencing]</p>
+
-
<p>Name: FA-6-28-1      Concentration: 4.3 ng/ul      A260/280: 1.79</p>
+
-
<p>Name: FA-6-28-2      Concentration: 4.9 ng/ul      A260/280: 2.00</p>
+
-
<p>Name: FA-6-28-3      Concentration: 7.3 ng/ul      A260/280: 1.64</p>
+
-
<p>2. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH5&alpha;), pCJDFB (DH5&alpha;) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.</p>
+
-
<p>3. Restriction enzyme digestion of D0-6-28-1, D0-6-28-2 and D0-6-28-3 with EcoR1 and Pst1.</p>
+
-
<p><img src="http://igem.org/wiki/images/b/b4/Zju_notebook1.jpg" width="80%"><p>
+
-
<p>It seems that we haven't achieved the correct plasmid but this may be caused by low concentration.</p>
+
-
<h3>June 29</h3>
+
-
<p>Amplify pCJDFA (DH5&alpha;) from bacteria of June 28.</p>
+
-
<h3>June 30</h3>
+
-
<p>1. Miniprep plasmid of pCJDFA (DH5&alpha;) from bacteria of June 29.</p>
+
-
<p>Name: FA-6-29-1      Concentration: 12.5 ng/ul      A260/280: 1.29  [Sequencing]</p>
+
-
<p>Name: FA-6-29-2      Concentration: 12.7 ng/ul      A260/280: 1.49  [Sequencing]</p>
+
-
<p>Name: FA-6-29-3      Concentration: 7.2 ng/ul        A260/280: 0.96</p>
+
-
<p>2. Amplify pCJDFB (DH5&alpha;) and pCJDFB (DH10&beta;) from bacteria of June28.</p>
+
-
<h3>July 01</h3>
+
-
<p>1. Receive correct sequencing results of D0-6-28-2 and D0-6-28-3.</p>
+
-
<p>2. Amplify bacteria of D0-6-28-2 and D0-6-28-3 for glycerol stock.</p>
+
-
<p>3. Miniprep plasmid of pCJDFB from bacteria of June 30.</p>
+
-
<p>Name: FB-7-01-1      Concentration: 7.1 ng/ul        A260/280: 2.20</p>
+
-
<p>Name: FB-7-01-2      Concentration: 25.8 ng/ul        A260/280: 2.05  </p>
+
-
<p>[Sequencing]</p>
+
-
<p>Name: FB-7-01-3      Concentration: 20.4 ng/ul        A260/280: 2.03</p>
+
-
<p>Name: FB-7-01-4      Concentration: 18.0 ng/ul        A260/280: 1.83  </p>
+
-
<p>[Sequencing]</p>
+
-
<h2>Week 3 (07.02-07.08): Co-transformation of pCJDD0, pCJDFA, pCJDFB</h2>
+
-
<h3>July 02</h3>
+
-
<p>1. Prepare LB plates of chloramphenicol resistance.</p>
+
-
<p>2. Sequencing of FA-6-29-2 and FA-6-29-2 failed.</p>
+
-
<h3>July 03</h3>
+
-
<p>1. Amplify pCJDFA (DH5&alpha;) from bacteria of June 29 for sequencing.</p>
+
-
<p>2. Co-transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3). Besides, transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3) respectively.</p>
+
-
<h3>July 04</h3>
+
-
<p>1. Pick up single colony of plate of D0 (BL21*(DE3)) of July 03 and amplify them in liquid LB medium.</p>
+
-
<p>2. Pick up single colony of plate of co-transformation of pCJDD0, pCJDFA, pCJDFB (BL21*(DE3)) of July 03 and amplify them on other LB plate.</p>
+
-
<h3>July 05</h3>
+
-
<p>1. Amplify co-transformation bacteria of July 04, FB bacteria of July 03 and E.coli BL21*(DE3) strain in liquid LB medium.</p>
+
-
<p>2. Make glycerol stock of D0 (BL21*(DE3)).</p>
+
-
<p>3. Transform pCJDFA into (BL21*(DE3) for transformation of July 03 failed.</p>
+
-
<h3>July 06</h3>
+
-
<p>1. Measure growth curve of D0 (BL21*(DE3)) and BL21*(DE3), respectively. We mistook the absorbance wavelength of spectrophotometer NanoDrop.</p>
+
-
<p>2. Pick up a single colony of plate of FA (BL21*(DE3)) of July 03 and amplify it in liquid LB medium.</p>
+
-
<p>3. Pick up a single colony of plate of co-transformation of July 04 and amplify it in liquid LB medium.</p>
+
-
<p>4. Amplify co-transformation bacteria of July 06 E.coli BL21*(DE3) strain in liquid LB medium for measurement of growth curve.</p>
+
-
<p>5. Make glycerol stock of FA (BL21*(DE3)), FB (BL21*(DE3)) and co-transformation (BL21*(DE3)).</p>
+
-
<p>6. Amplify backbone pSB1C3 by amplifying part in plate 4, 1E, 3E. Transform them into DH10&beta;.</p>
+
-
<h3>July 07</h3>
+
-
<p>1. Miniprep plasmid of pCJDFB from bacteria of July 06.</p>
+
-
<p>Name: FB-7-07          Concentration: 14.6 ng/ul        A260/280: 1.73</p>
+
-
<p>Name: Co-trans-7-07      Concentration: 9.7 ng/ul          A260/280: 1.79</p>
+
-
<p>2. Make the first part K738000, RNA scaffold with promoter and terminator. Do PCR procedure.</p>
+
-
<p><img src="http://igem.org/wiki/images/b/b0/Zju_notebook2.jpg" width="80%"><p>
+
-
<p>PCR of D0 seems to be correct, but there is nothing in pCJDFB and the plasmid of co-transformation bacteria is the same as that of pCJDD0.</p>
+
-
<p>3. Re-transformation:</p>
+
-
<p>No.  name  resistance  plate  strain</p>
+
-
<p>1  co-transform-2-1  A+K+S  Sp  BL21*DE3</p>
+
-
<p>2  co-transform-2-1  A+K+S  Kan  BL21*DE3</p>
+
-
<p>3  orginal FA  Sp  Sp  DH10&beta;</p>
+
-
<p>4  orginal FB  Kan  Kan  DH10&beta;</p>
+
-
<p>5  FA-6-30-1  Sp  Sp  DH10&beta;</p>
+
-
<p>6  FB-7-01-4  Kan  Kan  DH10&beta;</p>
+
-
<p>7  plate3 16H  Kan  Kan  BL21*DE3</p>
+
-
<p>When coated the plates, we made a mistake an put No.3 and No.4 bacteria together on Sp resistance plate, thus somehow leading to failure of these two transformations.</p>
+
-
<p>4. PCR purification of D0 PCR product.</p>
+
-
<p>5. Inoculate D0 bacteria in 3 resistance liquid LB medium to see if there is problem with antibiotics.</p>
+
-
<p>6. Double digest D0-PCR-product and pSB1C3 with EcoR1 and Pst1.</p>
+
-
<p>7. One of our teammate went to Peking and communicate with iGEM team there.</p>
+
-
<h3>July 08</h3>
+
-
<p>1. Measure growth curve: inoculate co-transformation 2-1 bacteria 1 ml into 50 ml liquid LB medium. Measurement was done with 723-Spectrum. It grew so slow that we gave up again.</p>
+
-
<p>2. Test the efficiency of competent cells with part in plate 1, 1G.</p>
+
-
<p>3. Amplify FA-7-3-1 and FA-7-3-2 bacteria for minipreparing plasmids later.</p>
+
-
<p>4. Miniprep plasmid of pCJDFB from bacteria of July 06.</p>
+
-
<p>Name: FA-7-8-1      Concentration: 54.1 ng/ul        A260/280: 1.88</p>
+
-
<p>Name: FA-7-8-2      Concentration: 45.1 ng/ul        A260/280: 1.89</p>
+
-
<p>The concentration is the highest among all pCJDFA we have miniprepared by now. However, its electrophoresis result is not correct at all.</p>
+
-
<p>5. Amplify glycerol stock bacteria FA (BL21*(DE3)) and FB (BL21*(DE3)) on plates.</p>
+
-
<p>6. Purify the products of double digestion of July 07.</p>
+
-
<h2>Week 4 (07.09-07.15): Make our own biobrick parts</h2>
+
-
<h3>July 09</h3>
+
-
<p>Prepare liquid SOB medium for co-transformation.</p>
+
-
<h3>July 10</h3>
+
-
<p>Amplify glycerol stock bacteria FB (DH5&alpha;) on plates.</p>
+
-
<h3>July 11</h3>
+
-
<p>1. Identify FA and FB with PCR.</p>
+
-
<p><img src="http://igem.org/wiki/images/a/a2/Zju_notebook3.jpg"  width="80%"><p>
+
-
<p>2. Amplify FB-7-10-plate bacteria in liquid LB medium.</p>
+
-
<h3>July 12</h3>
+
-
<p>1. Miniprep plasmid of pCJDFB from bacteria of July 06.</p>
+
-
<p>Name: FB-7-11      Concentration: 15.4 ng/ul        A260/280: 1.88</p>
+
-
<p>2. Amplify FB-7-10-plate1, FB-7-10-plate2, FA-6.29-liq (DH5&alpha;), FA-lix and FB-lix bacteria for minipreparing plasmids later.</p>
+
-
<p>3. Identify FB with PCR again.We found there were problems with primers used in those reactions, thus leading to the failure.</p>
+
-
<p><img src="http://igem.org/wiki/images/9/9f/Zju_notebook4.jpg"  width="80%"><p>
+
-
<p>4. Ligation: D0 and pSB1C3, purified at July 08.</p>
+
-
<h3>July 13</h3>
+
-
<p>1. Transformation:</p>
+
-
<p>No.name  time of heat shock/s</p>
+
-
<p>1  D0-FA-FB 90</p>
+
-
<p>2  FA-FB 90</p>
+
-
<p>3  D0-FA-FB 30</p>
+
-
<p>4  FA-FB 30</p>
+
-
<p>5  ligation product 90</p>
+
-
<p>6  D0 90</p>
+
-
<p>2. Miniprep plasmid of FA and FB from bacteria of July 12.</p>
+
-
<p>Name: FA-7-13-1-1      Concentration: 15.8 ng/ul        A260/280: 1.89</p>
+
-
<p>Name: FA-7-13-1-2      Concentration: 10.8 ng/ul        A260/280: 2.06</p>
+
-
<p>Name: FA-7-13-1-3      Concentration: 13.6 ng/ul        A260/280: 2.30</p>
+
-
<p>Name: FA-7-13-1-4      Concentration: 12.3 ng/ul        A260/280: 2.13</p>
+
-
<p>Name: FA-7-13-2-1      Concentration: 9.4 ng/ul          A260/280: 2.15</p>
+
-
<p>Name: FA-7-13-2-2      Concentration: 12.4 ng/ul        A260/280: 1.94</p>
+
-
<p>Name: FA-7-13-2-3      Concentration: 20.1 ng/ul        A260/280: 1.94</p>
+
-
<p>Name: FA-7-13-3-1      Concentration: 11.1 ng/ul        A260/280: 2.23</p>
+
-
<p>Name: FA-7-13-lix      Concentration: 13.3 ng/ul        A260/280: 2.08</p>
+
-
<p>Name: FB-7-13-yy      Concentration: 26.4 ng/ul        A260/280: 1.84</p>
+
-
<p><img src="http://igem.org/wiki/images/8/85/Zju_notebook5.jpg"  width="80%"><p>
+
-
 
+
-
<h3>July 14</h3>
+
-
<p>1. Co-transformation: </p>
+
-
<p>No.name  time of heat shock/s</p>
+
-
<p>1  D0-FA-FB  90</p>
+
-
<p>2  D0-FA-FB  30</p>
+
-
<p>Use SOC liquid medium.</p>
+
-
<p>2. Miniprep plasmid of FA and FB from bacteria of July 12.</p>
+
-
<p>Name: FB-7-14-1      Concentration: 42.9 ng/ul        A260/280: 0.67</p>
+
-
<p>Name: FB-7-14-2      Concentration: 21.0 ng/ul        A260/280: 2.69</p>
+
-
<p>Name: FB-7-14-3      Concentration: 44.7 ng/ul        A260/280: 0.59</p>
+
-
<p>Name: FB-7-14-4      Concentration: 14.4 ng/ul        A260/280: 2.09</p>
+
-
<p>Name: FB-7-14-5      Concentration: 33.1 ng/ul        A260/280: 0.68</p>
+
-
<p>Name: FB-7-14-6      Concentration: 26.1 ng/ul        A260/280: 1.64</p>
+
-
<p><img src="http://igem.org/wiki/images/3/3e/Zju_notebook6.jpg"  width="80%"><p>
+
-
<p>3. Amplify glycerol stock bacteria co-transformation (BL21*(DE3)) on 3 resistance plates, glycerol stock bacteria D0 on Amp plate.</p>
+
-
<p>4. Amplify FB-7-12-2, FB-7-12-4, FA-7-12-2-2 and FA-7-12-2-3 in LB liquid medium, named FB-7-14-2, FB-7-14-4, FA-7-14-2-2 and FA-7-14-2-3.</p>
+
-
<p>5. Digest FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with Pst1.</p>
+
-
<p><img src="http://igem.org/wiki/images/d/de/Zju_notebook7.jpg"  width="80%"><p>
+
-
<p>Maybe the concentration is too low that we can't recognize it.</p>
+
-
 
+
-
<h3>July 15</h3>
+
-
<p>1. Transformation:</p>
+
-
<p>No.  name time of heat shock/s  Strain</p>
+
-
<p>1  D0-FA-FB  90  DH10&beta;</p>
+
-
<p>2  FA-FB  90  DH10&beta;</p>
+
-
<p>3  D0-FA-FB  30  BL21*(DE3)</p>
+
-
<p>4  FA-FB  30  BL21*(DE3)</p>
+
-
<p>5  D0-pSB1C3  90  BL21*(DE3)</p>
+
-
<p>6  D0  90  BL21*(DE3)</p>
+
-
<p>2. Amplify bacteria in LB liquid medium:</p>
+
-
<p>No.  Name  From  quantity</p>
+
-
<p>1  Co-7-15  co-transformation (BL21*(DE3))  6</p>
+
-
<p>2  D0-7-15  D0-7-14  1</p>
+
-
<p>3  Co3-7-15-1-3-1  Co3-7-14-1  1</p>
+
-
<p>4  Co3-7-15-1-3-2  Co3-7-14-2  1</p>
+
-
<h2>Week 5 (07.16-07.22): Struggle to acquire correct plasmids</h2>
+
-
<h3>July 16</h3>
+
-
<p>1. Miniprep plasmid of FA and FB from bacteria of July 12.</p>
+
-
<p>Name: D0-7-16-1      Concentration: 60.9 ng/ul        A260/280: 1.65</p>
+
-
<p>Name: D0-7-16-2      Concentration: 34.0 ng/ul        A260/280: 1.93</p>
+
-
<p>Name: Penta          Concentration: 74.9 ng/ul          A260/280: 1.91</p>
+
-
<p>Name: RFP            Concentration: 201.2 ng/ul        A260/280: 1.90</p>
+
-
<p>2. Amplify glycerol stock D0 bacteria on two Amp plates, named D0-7-16-1 and D0-7-16-2.</p>
+
-
<p>3. Identify FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with PCR, new primers.</p>
+
-
<p><img src="http://igem.org/wiki/images/2/22/Zju_notebook8.jpg" width="80%"><p>
+
-
<p>The result of FA is not very ideal, while that of FB is not very exactly. The plasmid (all was Miniprep today) seems just stay where they were put in.</p>
+
-
<h3>July 17</h3>
+
-
<p>1. Amplify FA-FB-7-15-90 and FA-FB-7-15-30 in LB liquid medium.</p>
+
-
<p>2. Identify FA-7-13-2-2, FA-7-13-3-1 with PCR, increase Tm by 2Degrees Celsius.</p>
+
-
<p><img src="http://igem.org/wiki/images/5/5f/Zju_notebook9.jpg"  width="80%"><p>
+
-
<p>3. Amplify glycerol stock co-transformation-2-1 bacteria on plate, named co3-7-17.</p>
+
-
<h3>July 18</h3>
+
-
<p>Amplify FA-7-13-2-2, FA-7-13-2-3, FB-7-13-2 and FB-7-13-5 for minipreparing plasmids later.</p>
+
-
<h3>July 19</h3>
+
-
<p>1. Co-transformation of D0-7-16-2, FA-7-13-1-1 and FB-7-14-6, named Co3-7-18.</p>
+
-
<p>2. Amplify FB-7-12-1 and FB-7-12-3 in LB liquid medium.</p>
+
-
<p>3. Miniprep plasmid of FA-7-18-2-2 and FA-7-18-2-3.</p>
+
-
<p>Name: FA-7-18-2-2      Concentration: 17.4 ng/ul        A260/280: 2.19</p>
+
-
<p>Name: FA-7-18-2-3      Concentration: 14.7 ng/ul        A260/280: 2.27</p>
+
-
<h3>July 20</h3>
+
-
<p>Amplify glycerol stock Co3-2-1 bacteria on plate, named Co3-7-20.</p>
+
-
<h3>July 21</h3>
+
-
<p>1. Gradient PCR to find an appropriate Tm value for FA.</p>
+
-
<p><img src="http://igem.org/wiki/images/2/2e/Zju_notebook10.jpg"  width="80%"><p>
+
-
<p>It seems that there is not a lot of differences.</p>
+
-
<p>2. Amplify Co3-7-20 on plates, named Co3-7-21.</p>
+
-
<p>3. Amplify the following bacteria:</p>
+
-
<p>No  Name  Form  resistance  plate</p>
+
-
<p>1  Co2-7-21-1  Co2-7-17-1  K+S  K+S</p>
+
-
<p>2  Co2-7-21-2  Co2-7-17-2  K+S  K+S</p>
+
-
<p>3  Co2-7-21-3  Co2-7-17-3  K+S  K+S</p>
+
-
<p>4  control-kan  DH10&beta;  -  Kan</p>
+
-
<p>5  control-sp  DH10&beta;  -  Sp</p>
+
-
<p>6  control  DH10&beta;  -  -</p>
+
-
<h3>July 22</h3>
+
-
<p>1. Amplify Co3-7-21 in LB liquid medium, named Co3-7-22.</p>
+
-
<p>2. Colony PCR to test Co2-7-21, Co3-7-22, and FB-7-19.</p>
+
-
<p><img src="http://igem.org/wiki/images/a/a8/Zju_notebook11.jpg"  width="80%"><p>
+
-
<p>We found that we may use a wrong protocol of colony PCR.</p>
+
-
<p>3. Co-transform D0, FA and FB, named Co3-7-22. Co-transform FA and FB, named Co2-7-22.</p>
+
-
<p>4. Amplify FA-7-18-2-2, FA-7-18-2-3, FB-7-18-2 for minipreparing plasmids later.</p>
+
-
<p>5. Amplify D0-7-15-2 and Co3-7-21 on other plate, named D0-7-22-2 and Co3-7-22.</p>
+
-
<h2>Week 6 (07.23-07.29): Re-sequencing and re-test our original plasmids</h2>
+
-
<h3>July 23</h3>
+
-
<p>1. Amplify bacteria of FA-7-22 for glycerol stock.</p>
+
-
<p>2. Miniprep plasmid of FA-7-22-2-2 and FA-7-22-2-3.</p>
+
-
<p>Name: FA-7-22-2-2-1      Concentration: 20.1 ng/ul        A260/280: 2.19</p>
+
-
<p>Name: FA-7-22-2-2-2      Concentration: 15.3 ng/ul        A260/280: 2.05</p>
+
-
<p>Name: FA-7-22-2-3-1      Concentration: 24.2 ng/ul        A260/280: 1.91</p>
+
-
<p>Name: FA-7-22-2-3-2      Concentration: 22.3 ng/ul        A260/280: 1.69</p>
+
-
<p>3. Amplify glycerol stock bacteria FB on plates, named FB-7-23.</p>
+
-
<p>4. Amplify DH10&beta; on plate.</p>
+
-
<p>5. Amplify D0-7-23 plate in LB liquid medium.</p>
+
-
<p>6. Colony PCR of FA, FB and Co3.</p>
+
-
<p><img src="http://igem.org/wiki/images/1/1c/Zju_notebook12.jpg"  width="80%"><p>
+
-
<p>7. Miniprep plasmid of Co3-7-22-1 and Co3-7-22-2 for PCR procedure.</p>
+
-
<p>Name: Co3-7-23-1      Concentration: 29.9 ng/ul        A260/280: 1.91</p>
+
-
<p>Name: Co3-7-23-2      Concentration: 18.9 ng/ul        A260/280: 2.00</p>
+
-
<p>8. Amplify Co3-7-22-1, Co3-7-22-2, Co2-7-17-1, Co2-7-17-2 and Co2-7-17-3, named Co3-7-23-1, Co3-7-23-2, Co2-7-23-1, Co2-7-23-2 and Co2-7-23-3.</p>
+
-
<h3>July 24</h3>
+
-
<p>1. Miniprep plasmid of D0-7-23 and Co3-7-22-2 for PCR procedure.</p>
+
-
<p>Name: D0-7-24-2-1      Concentration: 40.6 ng/ul        A260/280: 2.00</p>
+
-
<p>Name: D0-7-24-2-2      Concentration: 43.7 ng/ul        A260/280: 1.98</p>
+
-
<p>Name: D0-7-24-2-3      Concentration: 42.3 ng/ul        A260/280: 2.03</p>
+
-
<p>Name: D0-7-24-2-4      Concentration: 51.7 ng/ul        A260/280: 1.87</p>
+
-
<p>Name: D0-7-24-2-5      Concentration: 40.7 ng/ul        A260/280: 1.87</p>
+
-
<p>Name: D0-7-24-2-6      Concentration: 28.9 ng/ul        A260/280: 1.83</p>
+
-
<p>Name: FB-7-24-2-1      Concentration: 18.2 ng/ul        A260/280: 2.32</p>
+
-
<p>Name: FB-7-24-2-4      Concentration: 16.6 ng/ul        A260/280: 1.95</p>
+
-
<p>Name: Co-7-24-3        Concentration: 27.1 ng/ul        A260/280: 1.84</p>
+
-
<p>Name: Co-7-24-9        Concentration: 15.3 ng/ul        A260/280: 1.81</p>
+
-
<p>Name: Co-7-24-10        Concentration: 27.0 ng/ul        A260/280: 1.78</p>
+
-
<p>Name: Co-7-24-12        Concentration: 15.5 ng/ul        A260/280: 1.77</p>
+
-
<p>2. Amplify FB-7-22-2 and FB-7-23 on other plates, named FB-7-24-2 and FB-7-24.</p>
+
-
<p>3. Transform FA-7-23-2-3-1 and FB-7-24-2-4 into BL21*(DE3), named Co2-7-24.</p>
+
-
<p>4. PCR to test (1) if pCJDD0 reacts with primer FAF and FAR; (2) further confirm the success of co-transformation.</p>
+
-
<p><img src="http://igem.org/wiki/images/c/c2/Zju_notebook13.jpg"  width="80%"><p>
+
-
<p><img src="http://igem.org/wiki/images/d/df/Zju_notebook14.jpg"  width="80%"><p>
+
-
<p>Unfortunately, we found we can achieve the same result of PCR whatever the template is if we use the same pare of primers. In contrast, we can't find there is any similarity between template and primers. We are really confused about that. It means that we can't test the success of co-transformation by PCR.</p>
+
-
<p>5. Amplify FB-7-23 bacteria in LB liquid medium.</p>
+
-
<h3>July 25</h3>
+
-
<p>Amplify Co-7-24-1-1, Co-7-24-1-2, Co-7-24-2-1 and Co-7-24-2-2, named Co-7-25-1-1, Co-7-25-1-2, Co-7-25-2-1 and Co-7-25-2-2. We prepared to induce them with IPTG later.</p>
+
-
<h3>July 26</h3>
+
-
<p>Transform original plasmids pCJDFA, pCJDFB and pCJDD0 into DH5&alpha; competent cells.</p>
+
-
<h3>July 27</h3>
+
-
<p>Peking iGEM_12 team visited us, we had a dinner together.</p>
+
-
<h3>July 28</h3>
+
-
<p>1. Peking and we ZJU introduced our projects this year briefly.</p>
+
-
<p>2. Amplify BL21(DE3)plyss strain on LB plates.</p>
+
-
<p>3. Transform FA-7-27-1, FA-7-27-2, FB-7-27-1 and FB-7-27-2 into DH5&alpha; competent cells respectively, named FA-7-28-1, FA-7-28-2, FB-7-28-1 and FB-7-28-2.</p>
+
-
<p>4. Ligation: D0 and pSB1C3, purified at July 08, named the product as K738000. Incubate at 22Degrees Celsius for 1h to increase the efficiency of transformation later.</p>
+
-
<p>5. Transform K738000 into DH5&alpha; competent cells, named K738000-7-28.</p>
+
-
<h3>July 29</h3>
+
-
<p>1. Amplify bacteria BL21(DE3)plyss in LB liquid medium for glycerol stock.</p>
+
-
<p>2. Amplify K738000-7-28 in LB liquid medium, named K738000-7-29.</p>
+
-
<h2>Week 7 (07.30-08.05): Test part BBa_K537009 and make our own parts</h2>
+
-
<h3>July 30</h3>
+
-
<p>1. Miniprep plasmid of K738000-7-29.</p>
+
-
<p>Name: K738000-7-30-1        Concentration: 60.0 ng/ul        A260/280: 1.86</p>
+
-
<p>Name: K738000-7-30-2        Concentration: 47.1 ng/ul        A260/280: 1.95</p>
+
-
<p>Name: K738000-7-30-3        Concentration: 90.1 ng/ul        A260/280: 1.95</p>
+
-
<p>Name: K738000-7-30-10      Concentration: 43.6 ng/ul        A260/280: 1.98</p>
+
-
<p>PCR of K738000-7-30</p>
+
-
<p><img src="http://igem.org/wiki/images/d/df/Zju_notebook15.jpg"  width="80%"><p>
+
-
<p>Unfortunately, there was a slight electrophoretic band in control. But the sequencing result came later verified that we've got the correct ligation product.</p>
+
-
<p>2. Pick up single colonies from plate of FB-7-26-2 and amplify it in LB liquid medium.</p>
+
-
<h3>July 31</h3>
+
-
<p>1. Amplify backbone pSB1C3:</p>
+
-
<p>Double digest K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.</p>
+
-
<p>2. Miniprep plasmid of K738000-7-29 and FA-7-28-1.</p>
+
-
<p>Name: K738000-7-30-4        Concentration: 83.9 ng/ul        A260/280: 1.94</p>
+
-
<p>Name: K738000-7-30-5        Concentration: 50.1 ng/ul        A260/280: 2.00</p>
+
-
<p>Name: K738000-7-30-11      Concentration: 46.0 ng/ul        A260/280: 1.97</p>
+
-
<p>Name: FA-7-28-1            Concentration: 33.0 ng/ul        A260/280: 2.04</p>
+
-
<p><img src="http://igem.org/wiki/images/e/e6/Zju_notebook16.jpg"  width="80%"><p>
+
-
<h3>August 01</h3>
+
-
<p>Double digest K738000-7-23-2 and K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.</p>
+
-
<h3>August 02</h3>
+
-
<p>1. Transform part K537009 into DH5&alpha; to acquire experience, named venus-8-2.</p>
+
-
<p>2. Transform original pCJDFA and pCJDFB into DH5&alpha;, named FA-8-2 and FB-8-2.</p>
+
-
<p>3. Transform part K398326 into DH10&beta; to amplify backbone pSB1C3 easier, BB-8-2.</p>
+
-
<h3>August 03</h3>
+
-
<p>1. Amplify BB-8-2, FA-8-2 and FB -8-2 for minipreparing plasmids later, named BB-8-3, FA-8-3 and FB-8-3.</p>
+
-
<h3>August 04</h3>
+
-
<p>1. Miniprep plasmid of BB-8-3 FA-8-3 and FB-8-3.</p>
+
-
<p>Name: BB-8-4        Concentration: 76.2 ng/ul        A260/280: 1.91</p>
+
-
<p>Name: BB-8-4        Concentration: 40.3 ng/ul        A260/280: 1.90</p>
+
-
<p>Name: FA-8-4        Concentration: 19.6 ng/ul        A260/280: 1.97</p>
+
-
<p>Name: FB-8-4        Concentration: 35.9 ng/ul        A260/280: 1.98</p>
+
-
<p><img src="http://igem.org/wiki/images/c/cc/Zju_notebook17.jpg"  width="80%"><p>
+
-
<p>Unfortunately, the PCR results were so different from that of original plasmids. We couldn't figure out the reason.</p>
+
-
<p>2. Amplify Venus-8-2 in LB liquid medium, named Venus-8-4.</p>
+
-
<p>3. Transform FAM-8-4-1, FAM-8-4-1, FBP-8-4-1 and FBP-8-4-1 into DH5&alpha;.</p>
+
-
<h3>August 05</h3>
+
-
<p>1. Pick up single colonies from plate of FAM-8-4 and FBP-8-4 and amplify it in LB liquid medium and LB plates.</p>
+
-
<p>2. Colony PCR of FAM-8-4-2 and FBP-8-4-2</p>
+
-
<p><img src="http://igem.org/wiki/images/c/c8/Zju_notebook18.jpg"  width="80%"><p>
+
-
<p>3. Double digest BB-8-4-1 with (1) EcoR1 and Pst1, (2) Xba1 and Spe1, get linear backbone through PCR cleanup kit.</p>
+
-
<p>4. Miniprep plasmid of Venus-8-4.</p>
+
-
<p>Name: Venus-8-5-1        Concentration: 168.1 ng/ul        A260/280: 1.91</p>
+
-
<p>Name: Venus-8-5-1        Concentration: 83.4 ng/ul          A260/280: 1.93</p>
+
-
<p>5. Pick up single colonies from plate of FAM-8-4-1, FAM-8-4-2, FBP-8-4-1 and FBP-8-4-2 and amplify it in LB liquid medium and LB plates. Miniprep plasmid of them.</p>
+
-
<p>Name: FAM-8-5-1        Concentration: 69.9 ng/ul          A260/280: 1.95</p>
+
-
<p>Name: FAM-8-5-2        Concentration: 57.3 ng/ul          A260/280: 1.95</p>
+
-
<p>Name: FAM-8-5-3        Concentration: 86.8 ng/ul          A260/280: 2.06</p>
+
-
<p>Name: FBP-8-5-2        Concentration: 21.0 ng/ul          A260/280: 2.14</p>
+
-
<p>Name: FBP-8-5-3        Concentration: 15.5 ng/ul          A260/280: 2.14</p>
+
-
<p>Name: FBP-8-5-5        Concentration: 15.6 ng/ul          A260/280: 2.07</p>
+
-
<p>6. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3). Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3).</p>
+
-
<h2>Week 8 (08.06-08.12): </h2>
+
-
<h3>August 06</h3>
+
-
<p>1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.</p>
+
-
<p>2. PCR of part K738001:</p>
+
-
<p><img src="http://igem.org/wiki/images/0/0a/Zju_notebook19.jpg"  width="80%"><p>
+
-
<p>3. PCR of original pCJDFA and pCJDFB.</p>
+
-
<p><img src="http://igem.org/wiki/images/f/f3/Zju_notebook20.jpg"  width="80%"><p>
+
-
<p>4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.</p>
+
-
<p>Name: FAM-8-6-1        Concentration: 56.7 ng/ul          A260/280: 1.95</p>
+
-
<p>Name: FAM-8-6-2        Concentration: 57.3 ng/ul          A260/280: 1.95</p>
+
-
<p>Name: FAM-8-6-3        Concentration: 86.8 ng/ul          A260/280: 2.06</p>
+
-
<p>Name: FBP-8-6-1        Concentration: 21.0 ng/ul          A260/280: 2.14</p>
+
-
<p>Name: FBP-8-6-2        Concentration: 15.5 ng/ul          A260/280: 2.14</p>
+
-
<p>Name: FBP-8-6-3        Concentration: 15.6 ng/ul          A260/280: 2.07</p>
+
-
<p>5. Add theophylline 0.08g/10mL into Venus-8-6, observe through fluorescence microscope.</p>
+
-
<p>6. Purify PCR product of K738001, named K738001-8-6.</p>
+
-
<p>7. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3), named Co3-8-6. Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3), named Co2-8-6.</p>
+
-
 
+
-
<h3>August 07</h3>
+
-
<p>1. Double digest K738001-8-6 with Xba1 and Spe1. Purify the product before ligation step. </p>
+
-
<p>2. Ligation: K738001-8-6 and pSB1C3.</p>
+
-
<p>3. Transform the product of ligation into DH5&alpha; competent cells.</p>
+
-
<h3>August 08</h3>
+
-
<p>Pick up single colonies from plate of K738001-8-7 and amplify it in LB liquid medium and LB plate.</p>
+
-
<h3>August 09</h3>
+
-
<p>1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium, induce with IPTG when OD600 is about 0.6 (mid log stage).</p>
+
-
<p>2. Miniprep plasmids of K738001-8-8:</p>
+
-
<p>Name: K738001-8-9-1        Concentration: 174.2 ng/ul          A260/280: 1.91</p>
+
-
<p>Name: K738001-8-9-2        Concentration: 298.5 ng/ul          A260/280: 1.87</p>
+
-
<p>3. Amplify FBP overnight for minipreparing plasmids later.</p>
+
-
<p>4. Mutation of D0: </p>
+
-
<p>PCR1: delete 3bp after MS2 in D0.</p>
+
-
<p>PCR2: delete 3bp before MS2 in D0.</p>
+
-
<p><img src="http://igem.org/wiki/images/9/94/Zju_notebook21.jpg"  width="80%"><p>
+
-
<p><img src="http://igem.org/wiki/images/b/bb/Zju_notebook22.jpg"  width="80%"><p>
+
-
<h3>August 10</h3>
+
-
<p>1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.</p>
+
-
<p>2. Miniprep plasmids of FB-8-9.</p>
+
-
<p>Name: FB-8-10-1        Concentration: 14.6 ng/ul          A260/280: 1.97</p>
+
-
<p>Name: FB-8-10-2        Concentration: 24.4 ng/ul          A260/280: 1.93</p>
+
-
<p>3. Mutation of D0: </p>
+
-
<p>PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.</p>
+
-
<p><img src="http://igem.org/wiki/images/7/7c/Zju_notebook23.jpg"  width="80%"><p>
+
-
<p>PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.</p>
+
-
<p><img src="http://igem.org/wiki/images/6/63/Zju_notebook24.jpg"  width="80%"><p>
+
-
<p>PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.</p>
+
-
<p><img src="http://igem.org/wiki/images/7/7d/Zju_notebook25.jpg"  width="80%"><p>
+
-
<p>4. Amplify FB-8-7 in LB liquid medium.</p>
+
-
 
+
-
<h3>August 11</h3>
+
-
<p>1. Miniprep plasmids of FB-8-11.</p>
+
-
<p>Name: FB-8-11-1        Concentration: 33.9 ng/ul          A260/280: 2.06</p>
+
-
<p>Name: FB-8-11-2        Concentration: 50.2 ng/ul          A260/280: 2.04</p>
+
-
<p>2. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Test fluorescence strength with hybrid synergy reader. Exciting light 535nm, absorbing light 480nm.</p>
+
-
 
+
-
<h3>August 12</h3>
+
-
 
+
-
 
+
-
<h2>Week 9 bogged, Subversion and start again</h2>
+
-
<h3>2012/8/13 Monday, August 13, 2012</h3>
+
-
<p>Yu Jianing and Yan came to Prof. Chen Xin for MD (molecular dynamics simulation, suggested by PKU iGem) modeling in RNA scaffold project. Prof. CHEN pointed out that it's so hard and costing for us green hand undergraduates to manipulate MD, which could be replaced by RNA prediction software and ODE modeling with matlab.</p>
+
-
 
+
-
<p>Zhang and Chen transform a group of D0, FA and FB with different combination.</p>
+
-
<p>LIU Xiao comes to carry out point mutation experiment to modify origin D0 in PCR method. He intended to build a library. However, we have only one month left, too short to finish the library.</p>
+
-
<p>Yan's fluorescent test with part K537009 is modestly successful. Most excitingly, Yan and Chen capture a love heart (or you can refer to it as a smiling smile) imaging by E.coli.</p>
+
-
<p><img src="http://igem.org/wiki/images/d/d2/Zju_notebook26.jpg"  width="80%"><p>
+
-
<h3>2012/8/14, Tuesday 14, 2012</h3>
+
-
<p>Chen makes competent cell with BL21 star DE3 borrow from Zhejiang Sci-Tech University.</p>
+
-
 
+
-
<h3>2012/8/15 Wednesday, August 15, 2012</h3>
+
-
<p>Prof. SHAO Jianzhong is willing to let us use the Synergy hybrid reader in his lab.</p>
+
-
<p>Chen and Zhang test fluorescence of D0, FA,FB,FA+D0,FA+FB,FA+FB+D0 with IPTG concentration gradient but find out that the fluorescence intensity are nearly no difference comparing with scaffold and without scaffold surprisingly.</p>
+
-
<h3>2012/8/16 Thursday, August 16, 2012</h3>
 
-
<p>After senior fellow's advises, however, we still can't see the result in < Organization of Intracellular Reactions with Rationally Designed RNA assemblies>.</p>
 
-
 
-
<p>Yan tried the parts of Theophylline riboswitch with YFP Venus reporter and it worked.
 
-
(Though excite 515nm emit 528 mn is too close for Synergy hybrid reader to detect and we change it into 505nm and 535nm.)</p>
 
-
<p><img src="http://igem.org/wiki/images/b/b4/Zju_notebook27.jpg"  width="80%"><p>
 
-
<p>You can see the fluorescence intensity increasing with Theophylline concentration.</p>
 
-
 
-
<p>All night talks and strange Sequencing results make us skeptical about the FAFB plasmids sent by French lab (which is crucial to our project). Maybe there's something wrong with our raw material.</p>
 
-
 
-
<h3>2012/8/17 Friday, August 17, 2012</h3>
 
-
<p>Today we faced mega-disaster that most of us thought the plasmids: FA & FB sent early August are not what we were asking for. Dr. Delebecque is so helpful and patient but he's away from his French lab now. He sent us an email providing a lot of information we need if we construct the plasmid on our own.
 
-
So we have to go back and start from the very beginning.
 
-
Chen, Zhang and Liu try to redesign the primers; Prof. Shao lent us the eGFP. Our advisor, Li Xin comes to discuss with us the technology of construct.</p>
 
-
 
-
<p>"You must be ready to burn yourself in your own flame: how could you become new, if you had not first become ashes?"
 
-
--When Nietzsche Wept</p>
 
-
 
-
<h3>2012/8/18 Saturday, August 18, 2012</h3>
 
-
<p>1 We bought commercial plasmids pCOLADuet?-1 Vector and pCDFDuet-1 Vector, and streak plate them respectively in Spectinomycin, Kanamycin and LB plate.</p>
 
-
<p>2 The part "2012 plate 1 15P" can't be transformed, as Zhang and Yan tried it twice.</p>
 
-
 
-
<h3>2012/8/19 Sunday, August 19, 2012</h3>
 
-
<p>1 plasmid miniprep:</p>
 
-
 
-
<p>Plasmid  Concentration  A260/280  A260/230</p>
 
-
<p>pCOLADuet?-1(1)  29.9 ng/&mu;L  1.96  1.21</p>
 
-
<p>pCOLADuet?-1(2)  25.6 ng/&mu;L  2.07  1.19</p>
 
-
<p>pCOLADuet?-1(3)  28.0 ng/&mu;L  2.07  1.15</p>
 
-
 
-
<p>2 LIU Xiao continues his point mutation experiment to modify origin D0 in PCR and build a library of changed RNA scaffold with different aptamer arm length. </p>
 
-
<p>He makes a new RNA scaffold, Nucleic Acid Electrophoresis as follows. </p>
 
-
<p>First step:</p>
 
-
<p><img src="http://igem.org/wiki/images/2/29/Zju_notebook28.jpg"  width="80%"><p>
 
-
<p>Second step:</p>
 
-
<p><img src="http://igem.org/wiki/images/7/7d/Zju_notebook29.jpg"  width="80%"><p>
 
-
<p>First step product is due in 50bp and second is due in 200bp.
 
-
But the Electrophoresis effect is not good.</p>
 
-
<p>3. Freshmen come to us and we taught them some methods for using Library Resources and making it in college.</p>
 
-
 
-
<h2>Week 10 keep moving on</h2>
 
-
<h3>2012/8/20 Monday, August 20, 2012</h3>
 
-
<p>1. Zhang continues LIU Xiao's point mutation D0 library experiment. According to the instructions of DNA gel extraction kit, she recover enzyme-digested products product pETDuet-BB, put new D0 into it.</p>
 
-
<p>2. We receive part K411003 and plate it on Chloramphenicol plates, and incubate.</p>
 
-
 
-
<h3>2012/8/21 Tuesday, August 21, 2012</h3>
 
-
<p>1. K411003 (Taipei 2010) were adding a series of Theophylline. This experiment is used both for testing parts and fishing for the condition of our synthetic D0 from Genescript's future GFP experiment. </p>
 
-
<p>2. The Synergy hybrid reader data is good. You can see the fluorescence intensity increasing with Theophylline concentration.</p>
 
-
<p><img src="http://igem.org/wiki/images/b/bb/Zju_notebook30.jpg"  width="80%"><p>
 
-
<h3>2012/8/22 Wednesday, August 22, 2012</h3>
 
-
<p>1. 00:39 in the morning, YU Jianing sends an e-mail to Yan from her hometown, attaching version 1 ODE modeling and some questions about biology meanings.</p>
 
-
<p>2. We start our major experiment: construct FA-2X-MS2 and FB-2X-PP7</p>
 
-
<p>2.1 Fragment</p>
 
-
<p>1. We PCR FA and FB from pEGFP. </p>
 
-
<p>2. We PCR MS2 from FAM( from Dr. Delebecque)</p>
 
-
<p>3. We PCR PP7 from FBP(from Dr. Delebecque )</p>
 
-
<p>Then use gel extraction kit to get these fragment.</p>
 
-
<p>2.2 Overlap PCR</p>
 
-
<p>First overlap PCR in our lab. We uses precisely designed primers to make FA and MS2 linked with 2X. So does FB and PP7.</p>
 
-
<p>3. Zhang goes on with LIU Xiao's point mutation and use Megaprimer PCR methods.</p>
 
-
 
-
<h3>2012/8/23 Thursday, August 23, 2012</h3>
 
-
<p>1. Yan PCR riboscaffold (synthesis by Genescript) and make it into Part.</p>
 
-
<p>2. Zhang continues mutation D0 and enzyme digest the fragment for PCR test.</p>
 
-
<p>3. LIU Huachun uses some parts form registry to construct a new EGFP for positive control.</p>
 
-
 
-
<h3>2012/8/24 Friday, August 24, 2012</h3>
 
-
<p>1. LIU reproduce pSB1C3</p>
 
-
<p>2. Yan continues to make part of riboscaffold.</p>
 
-
<p>3. Chen use Taq DNA Polymerase (ordinary) to put A onto FA-2X-MS2 and FB-2X-PP7 and link  it onto T vector.</p>
 
-
 
-
<h3>2012/8/25 Saturday, August 25, 2012</h3>
 
-
<p>1.  We extracted and amplified the mutation D0 library and then sent it to a sequencing facility</p>
 
-
<p>2.  LIU's pCDF-EGFP: transform into DH5&alpha; (It works pretty well.)</p>
 
-
<p>3.  Miniprep: Yan's last day's part riboscaffold</p>
 
-
 
-
<h3>2012/8/26 Sunday, August 26, 2012</h3>
 
-
<p>1.  Miniprep: pCDFDuet-EGFP and pColaDuet</p>
 
-
<p>2.  Part riboscaffold PCR: a little different from its original PCR from pETDuet Genescript.
 
-
So we should send sequencing for validation.</p>
 
-
 
-
<h2>Week 11 Recuperate</h2>
 
-
<h3>2012/8/27 Monday, August 27, 2012</h3>
 
-
<p>1.  Chen did the overlap PCR and construct FB-2X-PP7 (approximately 630 bp). It seems correct in the agarose gel electrophoresis. (1000 marker on the left and 5000 marker on the right)</p>
 
-
<p><img src="http://igem.org/wiki/images/2/24/Zju_notebook31.jpg"  width="80%"><p>
 
-
<p>2.  Yan and Chen PCR MS2 from plasmids Dr. Delebecque sent us respectively.
 
-
Yan uses 57.5 Degrees Celsius as melting temperature and Chen uses 58.5 and 63.5.
 
-
But the electrophoretic bands seem incorrect.
 
-
(Next day we find angrily that the primer the company synthesized is wrong!)</p>
 
-
<p><img src="http://igem.org/wiki/images/a/a1/Zju_notebook32.jpg"  width="80%"><p>
 
-
<p><img src="http://igem.org/wiki/images/9/95/Zju_notebook33.jpg"  width="80%"><p>
 
-
<h3>2012/8/28 Tuesday, August 28, 2012</h3>
 
-
<p>1.  LIU uses gel extraction to get last day Chen's MS2 and carry on overlap PCR to make FA-2X-MS2.</p>
 
-
<p>2.  Chen transforms FB-2X-PP7 (T vector) into Top 10.</p>
 
-
<p>3.  Repeated tests: Yan does the same as LIU and sets annealing temperature 59Degrees Celsius and 61Degrees Celsius. </p>
 
-
 
-
<h3>2012/8/29 Wednesday, August 29, 2012</h3>
 
-
<p>4.  Teacher Hu Yuhua from Edinburgh University interviews us concerning team collaboration. She offers that if we have oral English barriers we could come to her.</p>
 
-
<p>5.  LIU makes competent cell of DH10&beta;</p>
 
-
 
-
<h3>2012/8/30 Thursday, August 30, 2012</h3>
 
-
<p>All of us attend short semester and did nothing about igem today.</p>
 
-
 
-
<h3>2012/8/31 Friday, August 31, 2012</h3>
 
-
<p>1.  Sequencing result shows that FB-2X-PP7 we have constructed was correct in T vector.
 
-
So LIU prepare some for Miniprep and glycerol freeze store.</p>
 
-
<p>2.  Enzyme digestion and ligation: make FB-2X-PP7 from T vector into pColaDuet and transform it in DH5&alpha;.</p>
 
-
 
-
 
-
 
-
 
-
 
-
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Latest revision as of 03:17, 27 October 2012

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