Team:Hong Kong HKUST/wetlab/notebook

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<li><a href="#">Home</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor">Pneumosensor</a>
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<ul class="access-submenu">
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_one">Sensing</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_two">Expressing</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/parts">Parts</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/data">Data</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization">Characterization</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/results">Results</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/future_work">Future Work</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator">Riboregulator</a>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/CR_TA_Feature_Page">Feature Page</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/RNA_devices_catalog">Catalog Page</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/parts">Parts</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/data">Data</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">Characterization</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/results">Results</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/future_work">Future Work</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice">Human Practice</a>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit">Start-up Kit</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/handbook" >--Handbook</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/report" >--Report</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/database" >--Database</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/interview" >--Interview</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach">Outreach</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/Workshop" >--Workshop</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/talks" >--Talk</a></li>
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<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/isf_academy" >--ISF Academy</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/safety_and_ethics">Safety and Ethics</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team">Team</a>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/members">Members</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/advisors">Advisers</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/instructors">Instructors</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/attribution">Attributions</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/acknowledgement">Acknowledgement</a></li>
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<li><a href="https://igem.org/Team.cgi?year=2014">Official Team Profile</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab">Wetlab</a>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/notebook">Notebook</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a></li>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/safety">Safety</a></li>
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<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/Achievements">Achievements</a>
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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/medal_requirement">Medal Requirements</a></li>
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<h3 style="text-align:center">Notebook</h3>
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<h2>Notebook</h2>
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<p>
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Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and
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research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately.
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<h3>Pneumosensor</h3>
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<div id="satu" align="center"> <h2>June 2014</h2>
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      <div>
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      <a href="#" class="head">Week 1</a>
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      <div class="content" align="left" style="display: none;">
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<br><b>June 9</b> <br>
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Wet lab<br>
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·      Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600<br>
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·      Transformation of BBa_B0034 and BBa_E0040 into DH10B<br>
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<br><b>June 10</b> <br>
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Wet lab<br>
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·      Found reference promoter J23101 in BBa_I20260 from part registry<br>
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·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
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·      Bacterial inoculation of BBa_B0034 and BBa_E0040<br>
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<br><b>June 11</b> <br>
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Wet lab<br>
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·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
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·      Studied the Characteristic, function and mechnism of how promoters work<br>
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·      Miniprep of BBa_B0034 and BBa_E0040<br>
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<br><b>June 12</b> <br>
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Wet lab<br>
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·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
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·      research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of  p~<i>comE</i> to use for characterization<br>
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·      explain of scar<br>
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<br><b>June 13</b> <br>
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Wet lab<br>
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·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
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·      research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of  p~<i>comE</i> to use for characterization<br>
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      </div>
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    </div>
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<div>
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      <a href="#" class="head">Week 2</a>
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      <div class="content" align="left" style="display: none;">
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<br><b>June 16</b> <br>
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Wet lab<br>
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·      Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br>
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·      Found a possible P<sub>combox</sub><br>
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·      Transformation of BBa_J04450 and BBa_I20260 into DH10B<br>
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·      Bacterial inoculation of BBa_E0240<br>
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·   <br>
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<br><b>June 17</b> <br>
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Wet lab<br>
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·      Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br>
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·      Found a possible P<sub>combox</sub><br>
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·      Searched for other possible P<sub>combox</sub> sequences<br>
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·      Miniprep of BBa_E0240<br>
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·      Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br>
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·   <br>
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<br><b>June 18</b> <br>
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Wet lab<br>
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·      Designed primers for the P<sub>combox</sub> sequences<br>
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·      Received a P<sub>combox</sub> sequence from Wellcome Trust Sanger Institute<br>
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·      Miniprep of BBa_J04450(CHL) and BBa_I20260<br>
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·      Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br>
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·      Digestion of BBa_J04450(CHL) and BBa_I20260<br>
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·      Gel electrophoresis of digested products<br>
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·      <br>
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<br><b>June 19</b> <br>
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· Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI<br>
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· Miniprep of BBa_J04450(CHL) and BBa_I20260<br>
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· Digestion of BBa_J04450(CHL)<br>
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· Gel electrophoresis of digested product<br>
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· Bacterial inoculation of BBa_J04450(CHL)<br>
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· <br>
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<br><b>June 20</b> <br>
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· Amended primers of P<sub>combox</sub> sequences<br>
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· Miniprep of BBa_J04450(CHL)<br>
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· Digestion of BBa_J04450(CHL)<br>
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· Gel electrophoresis of digested product<br>
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· Designed primers for cloning <i>comD</i> and <i><i>comE</i></i><br>
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      </div>
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    </div>
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<div>
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      <a href="#" class="head">Week 3</a>
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      <div class="content" align="left" style="display: none;">
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<br><b>June 23</b> <br>
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Wet lab<br>
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·      Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br>
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·      Primer design for PCR of comX-myc and <i>comW</i>-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br>
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·      Designed plasmid construct for P<sub>combox</sub><br>
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·      Bacterial inoculation of BBa_J04450(CHL)<br>
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·   <br>
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<br><b>June 24</b> <br>
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Wet lab<br>
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·      Inoculation of BBa_B0015<br>
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·      Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br>
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·      Designed forward and reverse oligos of 67bp P<sub>combox</sub><br>
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·      Transformation of pSB1C3-BBa_I13504<br>
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·      Miniprep of BBa_J04450(CHL)<br>
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·      Transformation of BBa_J04450(KAN)<br>
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·      Bacterial inoculation of BBa_E0240<br>
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·   <br>
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<br><b>June 25</b> <br>
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Wet lab<br>
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·      Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br>
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·      Inoculation of BBa_K880005<br>
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·      Inoculation of pSB1C3-BBa_E0240<br>
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·      Miniprep of BBa_E0240<br>
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·      Digestion of BBa_E0240<br>
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·      Transformation of BBa_J04450(KAN)<br>
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·      Bacterial inoculation of BBa_J04450(CHL)<br>
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·      <br>
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·   <br>
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<br><b>June 26</b> <br>
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Wet lab<br>
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·      Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification<br>
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·      Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br>
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·      Gel electrophoresis of digested BBa_E0240<br>
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·      Gel extraction and purification of digested BBa_E0240 to obtain the insert<br>
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·      Miniprep of BBa_J04450(CHL)<br>
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·      Digestion of BBa_J04450(CHL)<br>
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·      Gel electrophoresis of digested BBa_J04450(CHL)<br>
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·      Gel extraction and purification of digested BBa_J04450(CHL) to obtain pSB1C3<br>
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·      Bacterial inoculation of BBa_K880005<br>
 +
·   <br>
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<br><b>June 27</b> <br>
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Wet lab<br>
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·      Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br>
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·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      Gel purification of pSB1C3-BBa_E0240<br>
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·      Miniprep of BBa_K880005<br>
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·   <br>
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      </div>
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    </div>
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<div>
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      <a href="#" class="head">Week 4</a>
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<br><b>June 30</b> <br>
 +
Wet lab<br>
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·      Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br>
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·      Gel check of purified BBa_B0015 insert<br>
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·      Transformation of ligated pSB1C3-K880005.B0015<br>
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·   <br>
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<div id="satu" align="center"> <h2><center>July 2014</center></h2>
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  <div>
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      <a href="#" class="head">Week 1</a>
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      <div class="content" align="left" style="display: none;">
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<br> <b> July 2</b> <br>
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Wet lab<br>
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·      Arrival of primers order for comX-myc and <i>comW</i>-FLAG<br>
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·      Dilution of primers and design of experiment for both comX-myc and <i>comW</i>-FLAG<br>
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·      Inoculation of ligated pSB1C3-K880005.B0015<br>
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·      Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br>
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·      Oligo annealing for PcomX<br>
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·      Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)<br>
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·      PCR for cloning <i>comD</i> and <i><i>comE</i></i><br>
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<br> <b> July 3</b> <br>
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Wet lab<br>
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·      PCR of comX-myc using Taq DNA Polymerase with ThermoPol Buffer to check whether primers are functional<br>
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·      Gel check for comX-myc PCR product<br>
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·      PCR of comX-myc using Phusion High-Fidelity PCR Master Mix with HF Buffer<br>
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·      Gel check for comX-myc PCR product<br>
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·      Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br>
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·      Received and stored the new common reverse primers for P<sub>combox</sub> and sense oligo and anti-sense oligo of 67bp P<sub>combox</sub><br>
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·      Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      Gel purification of digested BBa_E0240 and BBa_J04450<br>
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·   Inoculation of pSB3K3-BBa_J04450<br>
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·      PcomX Transformation<br>
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·      PcomCDE Oligo annealing, ligation, transformation<br>
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·      J04450 Plasmid Extraction<br>
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·      E0240:Plasmid extraction ; Digestion; Gel electrophoresis; Gel extraction ; Gel purification<br>
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·      B0015: Plasmid extraction ; Nanodrop<br>
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·      <i>comE<sup>D58E</sup></i>: PCR<br>
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·        <br>
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<br> <b> July 4</b> <br>
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Wet lab<br>
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·      PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br>
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·      Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      <i>comE<sup>D58E</sup></i> :Phusion PCR<br>
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·      E0240: Gel purification<br>
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·   <br>
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      </div>
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  </div>
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  <div>
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      <a href="#" class="head">Week 2</a>
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      <div class="content" align="left" style="display: none;">
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<br> <b> July 7</b> <br>
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Wet lab<br>
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·      Digestion of comX-myc with XbaI and PstI<br>
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·      Gel check of digested comX-myc, digestion clean up<br>
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·      PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br>
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·      Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br>
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·      Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br>
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·      Gel purification of digested BBa_E0240 and BBa_J04450<br>
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·      Submitted primers for Gibson assembly of pSB3K3-100bpP<sub>combox</sub><br>
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·      Inoculation of pSB3K3-BBa_J04450<br>
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·      E0240 - Bacterial inoculation<br>
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·      pSB1C3-PcomX: Bacterial inoculation<br>
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·      pSB1C3- PcomCDE: Bacterial inoculation, Streaking<br>
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·      <i>comE<sup>D58E</sup></i> :PCR cleanup; Digestion; Ligation with pSB1C3; Transformation<br>
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·   <br>
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<br> <b> July 8</b> <br>
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Wet lab<br>
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·      Ligation of comX-myc cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI<br>
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·      2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br>
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·      Transformation of ligation products<br>
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·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      Gel purification of digested BBa_J04450<br>
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·      Received pCEPcin from Universite de Toulouse, France<br>
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·      J04450 (KAN): Digestion; Gel electrophoresis; Gel extraction<br>
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·      E0240: Plasmid extraction<br>
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·      pSB1C3-PcomX: Plasmid extraction<br>
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·      pSB1C3- PcomCDE: Plasmid extraction<br>
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·      <i>comE<sup>D58E</sup></i>: Bacterial inoculation; Ligation; Transformation<br>
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·   <br>
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<br> <b> July 9</b> <br>
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Wet lab<br>
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·      Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br>
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·      Ethanol precipitation of pCEPcin<br>
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·      Gel purification of digested BBa_E0240<br>
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·      Gel check for BBa_E0240 insert and pSB3K3 backbone<br>
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·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Inoculation of pCEPcin<br>
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·      E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis<br>
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·      J04450 (KAN): Gel purification<br>
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·      <i>comE<sup>D58E</sup></i>:Plasmid extraction; NanoDrop; Gel electrophoresis (not <i>comE</i>); Bacterial inoculation<br>
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<br> <b> July 10</b> <br>
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Wet lab<br>
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·      Another PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br>
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·      Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br>
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·      Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br>
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·      Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br>
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·      Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI<br>
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·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
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·   Ethanol precipitation of BBa_E0240 insert<br>
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·      Ligate E0240-PcomX & PcomCDE <br>
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·      Extract J04450 (KAN)<br>
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·      Restrict check & run mutagenesis <i>comE<sup>D58E</sup></i><br>
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<br> <b> July 11</b> <br>
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Wet lab<br>
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·      Digestion of <i>comW</i>-FLAG with XbaI and PstI, digestion clean up<br>
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·      Gel check for the PCR product obtained on July 10<br>
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·      Digestion of pCEPcin at NcoI and XhoI<br>
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·      Ethanol precipitation of pCEPcin<br>
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·      Extract J04450 (KAN)<br>
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·      Ligate pSB1C3 with <i>comE<sup>D58E</sup></i> <br>
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·      TransformPcomCDE<br>
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·   <br>
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      </div>
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  </div>
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  <div>
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      <a href="#" class="head">Week 3</a>
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      <div class="content" align="left" style="display: none;">
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<br> <b> July 14</b> <br>
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Wet lab<br>
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·      Ligation of <i>comW</i>-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI<br>
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·      Transformation of ligation products<br>
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·      Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br>
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·      Digestion of pCEPcin at NcoI and XhoI<br>
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·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Gel purification of digested BBa_J04450<br>
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·      Inoculation of pCEPcin<br>
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·      PCR of <i>comE<sup>D58E</sup></i> with only 1 primer → check whether the primers are working or not<br>
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·      Ligation of pSB1C3-PcomX<br>
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·      Inoculation of pSB1C3-PcomCDE<br>
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·      Digest B0015<br>
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·      Digest <i>comE<sup>D58E</sup></i><br>
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·      Ligation of PCR product <i>comE</i> to pSB1C3<br>
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<br> <b> July 15</b> <br>
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Wet lab<br>
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·      Digestion check of ligated pSB1C3-K880005.comX-myc<br>
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·      Digestion of pSB1C3-K880005.comX-myc on SpeI and PstI, but no insert wanted present<br>
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·      PCR of comX using Vent DNA Polymerase, PCR clean-up<br>
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·      Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase<br>
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·      Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br>
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·      Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.<i>comW</i>-FLAG<br>
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·      Ethanol precipitation of pCEPcin<br>
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·      Gel check of the PCR product obtained on July 14<br>
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·   Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
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·      PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Plasmid extraction of pSB1C3-PcomCDE<br>
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·      Bacterial inoculation (5 for PcomCDE, 4 for PcomX)<br>
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·      Bacterial inoculation of B0015 (6 tubes)<br>
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·      Extract gel B0015 & <i>comE<sup>D58E</sup></i><br>
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·      K880005 dephosphorylation; Digestion<br>
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·   <br>
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<br> <b> July 16</b> <br>
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Wet lab<br>
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·      Digestion check of ligated pSB1C3-K880005.<i>comW</i>-FLAG<br>
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·      Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG<br>
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·      Gel check of pSB1C3-K880005.<i>comW</i>-FLAG PCR product (no PCR product present)<br>
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·      PCR of <i>comW</i> using Vent DNA Polymerase (no PCR product present)<br>
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·      Digestion of comX on XbaI and PstI<br>
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·      Gel check of the PCR product obtained on July 15<br>
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·      Gel purification of digested pSB3K3 backbone<br>
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·      Digestion of pCEPcin2 at XhoI and SalI<br>
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·   PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br>
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·   Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br>
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·      Plasmid extraction of Promoters and B0015<br>
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·      Colony PCR<br>
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·      Extract gel B0015 & <i>comE<sup>D58E</sup></i><br>
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·      Mutagenesis attempts for <i><i>comE</i></i><br>
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<br> <b> July 17</b> <br>
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Wet lab<br>
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·      PCR of comX-myc and comX using Vent DNA Polymerase<br>
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·      Gel check of comX-myc and comX PCR product, PCR clean-up<br>
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·      PCR of <i>comW</i> using Vent DNA Polymerase<br>
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·      Gel extraction and gel purification of <i>comW</i> PCR product<br>
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·      Dephosporylation of BBa_K880005<br>
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·      Gel check of the PCR product obtained on July 16, all types of P<sub>combox</sub> shown positive result<br>
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·      PCR clean-up of P<sub>combox</sub><br>
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·      PCR product ligation with pSB1C3<br>
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·      Bacterial inoculation of B0015, <i>comE<sup>D58E</sup></i><br>
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·      Extract gel B0015 & <i>comE<sup>D58E</sup></i> - Digestion<br>
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·      Overnight Ligation<br>
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·   <br>
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<br> <b> July 18</b> <br>
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Wet lab<br>
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·      PCR of comX using Vent DNA Polymerase<br>
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·      Gel check of comX PCR product, PCR clean-up<br>
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·      Digestion of comX PCR product on XbaI and PstI, digestion clean-up<br>
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·      Digestion of comX-myc and <i>comW</i> PCR products, digestion clean-up<br>
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·      Gel extraction and gel purification of digested comX and comX-myc<br>
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·      Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)<br>
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·      Digestion of pCEPcin2 (at BamHI and NcoI)<br>
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·   PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Gel check of the purified P<sub>combox</sub> obtained July 17<br>
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·   Gel check of the 100bp P<sub>combox</sub> (PCR product)<br>
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·      Gel purification of digested pSB3K3 backbone<br>
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·      Ligation of <i>comE<sup>D58E</sup></i><br>
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·      Gel extract <i>comE<sup>D58E</sup></i><br>
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·      BBa_B0015 Gel purification<br>
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·   <br>
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      </div>
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  </div>
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  <div>
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      <a href="#" class="head">Week 4</a>
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      <div class="content" align="left" style="display: none;">
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<br> <b> July 21</b> <br>
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Wet lab<br>
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·      Gel purification of comX and comX-myc<br>
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·      Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
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·      Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
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·      Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio<br>
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·      Ligation of <i>comW</i> into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
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·      Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG using Taq DNA Polymerase<br>
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·      PCR of 100bp and 150bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Gel purification of digested pSB3K3 backbone<br>
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·      Colony PCR of Promoters<br>
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<br> <b> July 22</b> <br>
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Wet lab<br>
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·      Colony PCR of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br>
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·      Inoculation of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG<br>
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·      Gel check of the PCR product obtained on July 21<br>
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·      Oligo annealing of 67bp P<sub>combox</sub><br>
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·      Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br>
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·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br>
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·      Gel electrophoresis of colony PCR results<br>
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·      Mutagenesis of <i>comE<sup>D58E</sup></i><br>
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·   <br>
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<br> <b> July 23</b> <br>
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Wet lab<br>
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·      Digestion check of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG colony PCR products on XbaI and SpeI<br>
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·      Inoculation of <i>comW</i>-FLAG<br>
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·      Gel check of PCR product obtained on July 22<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
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·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
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·      Gel electrophoresis of colony PCR results<br>
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·      Mutagenesis of <i>comE<sup>D58E</sup></i><br>
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·   <br>
 +
<br> <b> July 24</b> <br>
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Wet lab<br>
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·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and SpeI-HF<br>
 +
·      Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone<br>
 +
·      Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone<br>
 +
·      PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br>
 +
·      Digestion of comX-myc and comX on XbaI and PstI-HF<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel Check of PCR product obtained on July 23<br>
 +
·      Received pCEPcin2<br>
 +
·      Gel electrophoresis of colony PCR and mutagenesis results<br>
 +
·   <br>
-
<div id="flight">
+
<br> <b> July 25</b> <br>
-
<div id="satu" align="center"> <h1>June 2013</h1>
+
Wet lab<br>
-
    
+
·      Colony PCR of comX-myc using Taq DNA Polymerase<br>
-
       <div>
+
·      Gel check of comX-myc PCR product (no PCR product present)<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel check on K880005<br>
 +
·      Transformation of ligated product (promoters+pSB1C3)<br>
 +
·   <br>
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 5</a>
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      <div class="content" align="left" style="display: none;">
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<br> <b> July 28</b> <br>
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Wet lab<br>
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·      Colony PCR of comX-myc using Taq DNA Polymerase<br>
 +
·      Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br>
 +
·      Inoculation of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br>
 +
·      Colony PCR of promoters-pSB1C3<br>
 +
·      Bacterial inoculation of K880005, <i>comE<sup>D58E</sup></i> <br>
 +
·      <br>
 +
·   <br>
 +
 
 +
<br> <b> July 29</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Transformation of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      Inoculation of pSB3K3-BBa_J04450<br>
 +
·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Vent polymerase<br>
 +
·      Restrict check K880005 <br>
 +
·      Promoters colony PCR <br>
 +
·      PCR for <i>comE</i> fragments (mutagenesis) for Gibson Assembly<br>
 +
 
 +
 
 +
 
 +
<br> <b> July 30</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Inoculation of comX-myc and <i>comW</i>-FLAG <br>
 +
·      Streaking plates of comX-myc and <i>comW</i>-FLAG<br>
 +
·      PCR clean-up of 100bp,150bp and 160bp P<sub>combox</sub><br>
 +
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Restrict check K880005  <br>
 +
 
 +
<br> <b> July 31</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Redigest <i>comW</i>-FLAG on XmnI and NcoI<br>
 +
·      PCR sequencing for <i>comW</i> and comX<br>
 +
·      Ethanol precipitation for <i>comW</i> and comX<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      K880005 dephosphorylation & ligation<br>
 +
·      Promoters Colony PCR + Sequencing reaction<br>
 +
·      PCR for <i>comE<sup>D58E</sup></i> fragments (mutagenesis) for Gibson Assembly<br>
 +
 
 +
      </div>
 +
  </div>
 +
</div>
 +
<!------      AAAAAAAAAAAA    U        U  GGGGGGGGGGGG    U          U    SSSSSSSSSSSSS    TTTTTTTTTTTTTT      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                      T      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                  T      2 0 1 3---------------->
 +
<!------      A      A   U        U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      A      A   U     U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      AAAAAAAAAAAA   U        U  G              U          U    SSSSSSSSSSSSS          T        2 0 1 3---------------->
 +
<!------      A        A   U        U  G    GGGGGGG    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T 2 0 1 3---------------->
 +
<!------      A        A   U        U G          G    U          U                S        T 2 0 1 3---------------->
 +
<!------      A        A   UUUUUUUUUUU  GGGGGGGGGGGG    UUUUUUUUUUUUU    SSSSSSSSSSSSS        T        2 0 1 3---------------->
 +
 
 +
 
 +
<div id="satu" align="center"> <h2>August 2014</h2>
 +
   <div>
 +
       <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 1</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-BBa_J04450<br>
 +
·      Restrict check Promoters & Sequencing reaction<br>
 +
·      Transform K880005<br>
 +
·   <br>
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 4</b> <br>
 +
Wet lab<br>
 +
·      Digestion of comX-myc on EcoRI-HF and SpeI, gel extraction and gel purification<br>
 +
·      Ligation of BBa_B0015 cut on XbaI and PstI into pSB1C3-comX-myc backbone cut on SpeI and PstI<br>
 +
·      Transformation of pSB1C3-comX-myc.B0015 ligation product<br>
 +
·      Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product<br>
 +
·      Sequencing of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Inoculation of comX-myc<br>
 +
·      Digestion of 100bp, 150bp, 160bp and 180bp P<sub>combox</sub> at XbaI and PstI<br>
 +
·      Ligation of 100bp and 160bp P<sub>combox</sub> with pSB1C3<br>
 +
·   Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      PcomCDE Precipitation<br>
 +
·      PcomX Sequencing reaction & precipitation<br>
 +
·      Transform <i>comE<sup>D58E</sup></i>  <br>
 +
·      Gibson Assembly for <i>comE</i> and <i>comE<sup>D58E</sup></i> fragments<br>
 +
 
 +
<br><b>Aug 5</b> <br>
 +
Wet lab<br>
 +
·      Sequencing result failed<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product failed<br>
 +
·      Digestion check of comX-myc on StyI<br>
 +
·      Colony PCR of comX-myc<br>
 +
·      Inoculation of comX-myc<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Ligation of 150bp, 180bp and 249bp P<sub>combox</sub> with pSB1C3<br>
 +
·      Transformation of pSB1C3-150bp P<sub>combox</sub> , pSB1C3-180bp P<sub>combox</sub>  and pSB1C3-249bp P<sub>combox</sub><br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·   Inoculation of pCEPcin2<br>
 +
·      Send to sequencing: PcomCDE and PcomX<br>
 +
·      Bacterial inoculation of <i>comE<sup>D58E</sup></i>  <br>
 +
·      Colony PCR to check pSB1C3-<i><i>comE</i></i> and pSB1C3-<i><i>comE</i><sup>D58E</sup></i><br>
 +
 
 +
 
 +
<br><b>Aug 6</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc on XbaI and PstI<br>
 +
·      Gel check of comX-myc PCR product (no PCR product present)<br>
 +
·      Sequencing of <i>comW</i>-FLAG using VF2 and VR<br>
 +
·      Digestion of comX-myc on StyI<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI<br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Phosphorylation of 67bp P<sub>combox</sub> oligos<br>
 +
·      Oligo annealing for 67bp P<sub>combox</sub><br>
 +
·      Align sequence with expected: P<sub>comCDE</sub> and P<sub>comX</sub><br>
 +
 
 +
 
 +
<br><b>Aug 7</b> <br>
 +
Wet lab<br>
 +
·      Digestion of comX-myc on StyI<br>
 +
·      Digestion check of comX on SphI and NcoI<br>
 +
·      Digestion check of <i>comW</i> on XmnI and NcoI<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·      Precipitate after sequencing reaction PcomCDE<br>
 +
·      Make glycerol stock for PcomX<br>
 +
·      Run colony PCR for <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
 +
<br><b>Aug 8</b> <br>
 +
Wet lab<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification<br>
 +
·      Digestion of <i>comW</i>-FLAG on SpeI and PstI-HF, digestion clean-up<br>
 +
·      Sequencing of comX-myc succeeded but mutation at EcoRI site<br>
 +
·      Digestion check of comX-myc at EcoRI-HF and PstI-HF<br>
 +
·      Ligation of comX and <i>comW</i> into dephosporylated pSB1C3-K880005<br>
 +
·      Transformation of comX and <i>comW</i> ligation product<br>
 +
·      Digestion of comX-myc at EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br>
 +
·      Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> with pSB1C3 backbone<br>
 +
·      Ligate with E0240 PcomX  <br>
 +
·      Colony PCR for <i>comE</i> mutant<br>
 +
·      Check sequence PcomCDE<br>
 +
·   <br>
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 11</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Gel purification of comX-myc digested on EcoRI-HF and SpeI<br>
 +
·      Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 180bp and 249bp) at XbaI and PstI<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp)<br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·   Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Extract pSB1C3 from J04450<br>
 +
·      Redo ligation part 1<br>
 +
·      Gibson assembly of <i><i>comE</i><sup>D58E</sup></i><br>
 +
·      Ligation of PCR product <i>comD</i> to pSB1C3<br>
 +
 
 +
<br><b>Aug 12</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Dilution of <i>comW</i>-FLAG<br>
 +
·      Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
 +
·      Re-ligation of pSB1C3-B0015.comX-myc<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product<br>
 +
·      Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
 +
·      Ligate K880005 and <i>comE</i>(D58E)<br>
 +
·      Inoculate for glycerol stock (PcomX)<br>
 +
·   <br>
 +
<br><b>Aug 13</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Inoculation of  pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      Check new dNTP<br>
 +
·      Restrict check of ligation product part 1<br>
 +
·      Colony PCR for checking part 2 ligation product and gibson assembly<br>
 +
·      Inoculation of PcomX for glycerol stock<br>
 +
·      <br>
 +
·   <br>
 +
<br><b>Aug 14</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Transformation of ligated product pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Dilution of <i>comW</i>-FLAG<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Ligation of <i>comW</i>-FLAG into pSB1C3-B0015 backbone<br>
 +
·      Re-digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Gel check of the digested pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13<br>
 +
·      Colony PCR of Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      glycerol stock for PcomX<br>
 +
·      colony PCR ligation product part 1<br>
 +
·      colony PCR gibson assembly<br>
 +
·      Run gel for PCR products<br>
 +
·      Inoculation of <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
 +
<br><b>Aug 15</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br>
 +
·      Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Overnight ligation of BBa_B0015 into pSB1C3-K880005.comX-myc backbone at 16°C<br>
 +
·      Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product<br>
 +
·      Ligation of comX-myc into pSB1C3-B0015 backbone<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Preparation for submittion of samples to be sequenced<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      colony PCR for ligation product part 1<br>
 +
·      colony PCR for ligation product part 2<br>
 +
·      colony PCR for gibson assembly<br>
 +
·      Run gel for PCR products<br>
 +
·      <i>comE</i>-D58E plasmid extraction<br>
 +
·   <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
       <a href="#" class="head">Week 4</a>
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>June 24</b><br>
+
<br><b>Aug 18</b> <br>
-
Wet lab <br>
+
Wet lab<br>
-
·      Inoculation of pBlueScript KS(+) for training <br>
+
·      Transformation of ligated pSB1C3-K880005.comX-myc<br>
-
·      Autoclave basic materials <br>
+
·      Ligation of <i>comW</i>-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone<br>
-
·      Preparing LB <br>
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
-
·      LB-Ampicillin plates poured
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on SpeI-HF and PstI-HF<br>
-
Dry lab <br>
+
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
-
Protocols review <br>
+
·      Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Sent out pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp) samples for commercial sequencing<br>
 +
·   Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at SpeI and PstI<br>
 +
·      Gel purification of digested pSB1C3-P<sub>combox</sub> (150bp, 160bp, 180bp and 249bp)<br>
 +
·   Inoculation of pSB1C3-BBa_E0240<br>
 +
·      Gel extraction and purification of E0240<br>
 +
·      Bacterial inoculation of PcomX (PcomCDE(3))<br>
 +
·      Gel extraction and purification of <i>comE</i> mutant<br>
 +
·   <br>
-
<br><b>
+
<br><b>Aug 19</b> <br>
-
June 25 </b><br>
+
Wet lab<br>
-
Wet lab <br>
+
·     Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
-
· Plasmid extraction of pBlueScript KS(+)  <br>
+
·      Digestion check of comX-myc on XbaI and PstI-HF<br>
-
·      LB-Chloramphenicol plates poured <br>
+
·      Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
-
Dry lab <br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br>
-
·      Primers design for FABP1 promoter, PPAR-alpha promoter, GRP78 promoter <br>
+
·      Gel purification of digested pSB1C3-P<sub>combox</sub> (100bp)<br>
-
·      Informal meeting <br>
+
·      Ligation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) with BBa_E0240 insert<br>
 +
·      Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·      Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br>
 +
·      Gel purification of E0240 insert<br>
 +
·      Digest PcomX(PcomCDE(3))-pSB1C3<br>
 +
·      Make aliquotes for oligo - PcomCDE<br>
 +
·      PcomCDE Oligo annealing<br>
 +
·      Ligation of <i>comE</i> mutant insert with K880005<br>
 +
·      Ligation of <i>comE</i> insert with K880005<br>
 +
·      Gel electrophoresis of Gibson assembly mastermix checking<br>
 +
·      Plasmid extraction of J04450(CHL), PcomX(PcomCDE(3)) <br>
 +
·      Bacterial inoculation of <i>comE</i> WT, J04450 (CHL)<br>
 +
·   <br>
 +
<br><b>Aug 20</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI<br>
 +
·      Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Gel purification of digested pSB1C3-BBa_E0240<br>
 +
·      Digestion of pSB1C3-180bp P<sub>combox</sub> at SpeI and PstI<br>
 +
·      Digestion clean-up of pSB1C3-180bp P<sub>combox</sub><br>
 +
·      Ligation of pSB1C3-180bp P<sub>combox</sub> with BBa_E0240 insert<br>
 +
·      Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·      Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·   Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·      Ligation of E0240 insert with PcomX<br>
 +
·      Transformation [E0240-PcomCDE exp+ -ve, pSB1C3-PcomCDE exp+ -ve]<br>
 +
·      Ligation of <i>comE<sup>D58E</sup></i> insert with K880005<br>
 +
·      Gel extract <i>comE</i> WT<br>
 +
·      PCR check for <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Digest J04450(CHL) Gel extraction<br>
 +
·      Plasmid extraction of <i>comE</i> WT, J04450 (CHL)<br>
 +
·   <br>
 +
<br><b>Aug 21</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
 +
·      Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase<br>
 +
·      Digestion of <i>comW</i>-FLAG on NotI-HF<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Transformation pSB1C3-K880005.<i>comW</i>-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) at NcoI<br>
 +
·      Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·   Received sequencing result of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp)<br>
 +
·      Ligate PcomX with E0240 insert<br>
 +
·      PCR, Restrict check Part I (PcomCDE)<br>
 +
·      O/N Ligation w/ PcomCDE<br>
 +
·      Extract <i>comE</i> WT-pSB1C3<br>
 +
·      PCR and restrict check <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Glycerol stock for PcomX <br>
 +
·   <br>
 +
<br><b>Aug 22</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF<br>
 +
·      Inoculation pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br>
 +
·      PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) using Vent polymerase <br>
 +
·      Sent out pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) for commercial sequencing<br>
 +
·      Transformation of newly ligated (E0240)<br>
 +
·      Bacterial inoculation of checked colony (E0240)<br>
 +
·      O/N ligation w/ pSB1C3<br>
 +
·      Ligation <i>comE</i> WT-pSB1C3<br>
 +
·      Ligate <i>comE<sup>D58E</sup></i>-myc w/ K880005<br>
 +
·   <br>
-
<br><b>
 
-
June 26 </b><br>
 
-
Wet lab <br>
 
-
·      Extraction of gDNA from HepG2 Cells for FABP1 promoter and PPAR-alpha promoter <br>
 
-
·      Transformation of pEGFP-N1 and <a href="http://parts.igem.org/Part:BBa_K817002">BBa_K817002</a> (P<i>fadBA</i>) <br>
 
-
<br><b>
 
-
June 27 </b><br>
 
-
Wet lab <br>
 
-
·      Inoculation of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) for plasmid extraction <br>
 
-
<br><b>
+
      </div>
-
June 28</b> <br>
+
    </div>
-
Wet lab <br>
+
<div>
-
·      Extraction of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) plasmids by miniprep <br>
+
      <a href="#" class="head">Week 5</a>
-
·      Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification <br>
+
      <div class="content" align="left" style="display: none;">
-
·      Restriction of c(P<i>fadBA</i>) by EcoR1 and Pst1 <br>
+
<br><b>Aug 25</b> <br>
-
·      Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (P<i>fadBA</i>) <br>
+
Wet lab<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
 +
Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
 +
Transformation of pSB1C3-K880005.<i>comW</i>-FLAG
 +
Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, gel extraction and gel purification
 +
Inoculation of pSB1C3-K880005.comX-myc.B0015 for glycerol stock<br>
 +
·      Received sequencing result of pSB1C3-P<sub>combox</sub> (160bp and 249bp)<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br>
 +
·      Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 160bp, 180bp and 249bp) at XbaI and PstI<br>
 +
·      Gel purification of digested P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) insert<br>
 +
·      Ligation of PcomX-E0240<br>
 +
·      Digestion of PcomCDE-E0240<br>
 +
·      Colony PCR pSB1C3-PcomCDE, K880005-<i>comE</i> WT, K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
 +
<br><b>Aug 26</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF<br>
 +
·      Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br>
 +
·      Gel pSB1C3-PcomCDE<br>
 +
·      PcomX-E0240 Colony PCR check<br>
 +
·      PcomCDE glycerol stock <br>
 +
·      Restrict check K880005-<i>comE</i> WT<br>
 +
·      Ligation w/ <i>comE</i> WT<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
 
 +
·   <br>
 +
<br><b>Aug 27</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Sequencing of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone <br>
 +
·      Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br>
 +
·      Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 180bp and 249bp), pSB1C3-P<sub>combox</sub> (160bp)<br>
 +
·      pSB1C3-PcomCDE<br>
 +
·      PcomCDE sequencing <br>
 +
·      PCR check K880005-<i>comE</i> WT<br>
 +
·      PCR check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Ligate K880005-<i>comE</i>-myc<br>
 +
 
 +
·   <br>
 +
<br><b>Aug 28</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br>
 +
·      Colony PCR of pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      pSB1C3-PcomCDE overnight ligation<br>
 +
·      PcomX-E0240 ligation<br>
 +
·      Gel extract I20260 and E0240<br>
 +
·      Glycerol stock for K880005-<i>comE</i> WT<br>
 +
·      Colony PCR for K880005-<i>comE<sup>D58E</sup></i>-myc  <br>
 +
·      Ligate K880005-<i>comE</i>-myc <br>
 +
·      PCR <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Ligation of Part 1 and Part 2<br>
 +
 
 +
·   <br>
 +
<br><b>Aug 29</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Sent out pSB1C3-160bp P<sub>combox</sub> for commercial sequencing<br>
 +
·      Digestion of pSB1C3-160bp P<sub>combox</sub> at SpeI and PstI<br>
 +
·      Colony PCR for pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Ligation of pSB1C3-160bp P<sub>combox</sub> with BBa_E0240 insert<br>
 +
·      Transform pSB1C3-PcomCDE <br>
 +
·      Transform PcomX-E0240 <br>
 +
·      Ligate I20260 and E0240 <br>
 +
·      Ligate K880005-<i>comE</i> WT  <br>
 +
·      Ligate K880005-<i>comE</i>-myc<br>
 +
·      Ligate K880005-<i>comE<sup>D58E</sup></i>-myc  <br>
 +
·      Gel check <i>comE<sup>D58E</sup></i>-myc <br>
 +
·   <br>
-
<br><b>
 
-
June 30 </b><br>
 
-
Dry lab <br>
 
-
Experiment planning and protocols revision for next week work <br><br>
 
       </div>
       </div>
 +
    </div>
 +
 +
       </div>
       </div>
 +
    </div>
 +
<!---- SSSSSSSS  EEEEEEEE  PPPPPPPP  TTTTTTTTTTTT  EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  RRRRRRRR    2 0 1 3-------->
 +
<!---- S          E          P      P      T        E          MM      MM  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M M    M M  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M  M  M  M  B      B  E          R  R 2 0 1 3-------->
 +
<!----     S          E          P      P        T        E          M  M M  M  B      B  E          R    R 2 0 1 3-------->
 +
<!----      SSSSSSSS  EEEEEEEE  PPPPPPPP        T        EEEEEEEE  M    M    M  BBBBBBBB    EEEEEEEEE  RRRRRRRR 2 0 1 3-------->
 +
<!----            S  E          P              T        E          M        M  B      B  E          R  R      2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R  2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R 2 0 1 3-------->
 +
<!----   S  E          p              T        E          M        M  B      B  E          R      R 2 0 1 3-------->
 +
<!---- SSSSSSSS  EEEEEEEE  P              T        EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  R  R 2 0 1 3-------->
-
</div>
+
<div id="satu" align="center"> <h2>September 2014</h2>
-
</div>
+
-
<div id="satu" align="center"> <h1><center>July 2013</center></h1>
+
    
    
-
  <div>
+
<div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br> <b> July 2 </b> <br>
+
<br><b>Sept 1</b> <br>
-
Wet lab <br>
+
Wet lab<br>
-
·      Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br>
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br>
-
·      LB-Chloramphenicol plates (the previous ones were found contaminated) <br>
+
·      Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI<br>
-
·      Inoculation of pEGFP-N1 for plasmid extraction <br>
+
·      Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br>
-
·      Transformation of P<i>fadBA</i> due chloramphenicol plates contamination <br>
+
·      Received the sequencing result of pSB1C3-160bp P<sub>combox</sub><br>
 +
·      Colony PCR: pSB1C3-PcomCDE, PcomX-E0240, I20260+Part2, E0240+Part2, E0240+Part2, K880005-<i>comE</i> WT, K990005-<i>comE</i>-myc, K990005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
-
<br> <b> July 3 </b> <br>
+
<br><b>Sept 2</b> <br>
-
·      Extraction of pEGFP-N1 plasmids by miniprep <br>
+
Wet lab<br>
-
·      PCR for PPAR-alpha promoter amplification <br>
+
·      Ligation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP cut on EcoRI-HF and PstI-HF into pSB3K3 cut on EcoRI-HF and PstI-HF<br>
-
·      Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br>
+
·      Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br>
 +
·      Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240 at XmnI and NcoI<br>
 +
·      Whole construct gel check <br>
 +
·      Restrict check PcomX-E0240<br>
 +
·      Restrict check Part 1 + Part 2<br>
 +
·      Restrict check I20260 and E0240 <br>
 +
·      Restrict check K880005-<i>comE</i> WT<br>
 +
·      Restrict check K880005-<i>comE</i>-myc<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
-
<br> <b> July 4</b> <br>
+
<br><b>Sept 3</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Inoculation of BBa_K817002  P<i>fadBA</i><br>
+
·      Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br>
-
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
-
·      Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78<br>
+
·      Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Restrict check I20260-Part 2<br>
 +
·      Restrict check E0240-Part 2<br>
 +
·      Restrict check Whole construct<br>
 +
·      Restrict check -ve of whole construct ligation<br>
 +
·      Restrict check pSB1C3-PcomCDE<br>
 +
·   <br>
-
<br> <b> July 5</b> <br>
+
<br><b>Sept 4</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Extraction of BBa_K817002 (P<i>fadBA</i>) <br>
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br>
-
·      Inoculation of pEGFP-N1<br>
+
·      Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br>
-
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
·      Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF<br>
-
·      Gel check, 0.8% gel for previously gDNA extraction<br>
+
·      Transformation of pSB3K3-Combox.GFP ligation product<br>
-
·      Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter<br>
+
·      Digestion of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br>
-
·      Poured new LB-Kanamycin plates<br>
+
·      Ligation of 160bp P<sub>combox</sub>-BBa_E0240,comX and pSB3K3<br>
-
·      Transformation of <a href="http://parts.igem.org/Part:BBa_J176171">BBa_J176171</a><br>
+
·      Restrict check I20260-Part 2, E0240-Part 2, I20260-Part 2, whole construct, -ve of whole construct ligation<br>
-
Dry lab<br>
+
·      Restrict check PcomCDE-E0240<br>
-
·      Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector<br>
+
·      Restrict check I20260, E0240, Whole construct 3,4,5<br>
-
·      Primer redesign for PPAR-alpha promoter and FABP1 promoter<br><br>
+
·      Restrict check and colony PCR for pSB1C3-PcomCDE<br>
 +
·      Restrict check K880005+<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Restrict check  K880005+<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 5</b> <br>
 +
Wet lab<br>
 +
·      Inoculation of pSB3K3-Combox.GFP<br>
 +
·      Sequencing for pSB1C3-PcomCDE<br>
 +
·      <br>
 +
·   <br>
       </div>
       </div>
-
  </div>
+
    </div>
-
 
+
<div>
-
  <div>
+
       <a href="#" class="head">Week 2</a>
       <a href="#" class="head">Week 2</a>
-
       <div class="content" align="left">
+
       <div class="content" align="left" style="display: none;">
-
<br> <b> July 8</b> <br>
+
<br><b>Sept 8</b> <br>
Wet lab<br>
Wet lab<br>
-
·      gDNA extraction from HepG2 cells<br>
+
·      Digestion check of pSB3K3-Combox.GFP on XmnI<br>
-
·      Restriction of BBa_K817002 P<i>fadBA</i><br>
+
·      Restrict check and plasmid PCR for PcomCDE-E0240<br>
-
·      Gel check for gDNA extraction<br>
+
·      Obtain sequencing results for PcomCDE-pSB1C3, whole construct (PcomX)<br>
-
·      Inoculation of BBa_J176171, BBa_K817002 (P<i>fadBA</i>), pEGFP-N1 for plasmid extraction<br>
+
·      K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc digest<br>
-
·      New primers for PPAR-alpha promoter and FABP1 promoter arrival<br>
+
·      Ligation of Part 1 and 2 (also I20260, E0240)<br>
-
·      Ran gel for BBa_K817002 P<i>fadBA</i> restriction check, gel extraction and purification<br>
+
·      Transformation<br>
 +
 
 +
·   <br>
 +
 
 +
 
 +
<br><b>Sept 9</b> <br>
 +
Wet lab<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 10</b> <br>
 +
Wet lab<br>
 +
·      Streak plate for pSB3K3-Combox.GFP<br>
 +
·      Restrict check PcomCDE-E0240<br>
 +
·      Obtain sequencing results for PcomCDE-pSB1C3<br>
 +
·      Whole construct (PcomX)<br>
 +
·      K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc send to sequencing<br>
 +
·      Ligation and transformation of Part 1 and 2 (also I20260, E0240)<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 11</b> <br>
 +
Wet lab<br>
 +
·      Restriction check of pSB3K3-comX-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br>
 +
·      Obtain sequencing results PcomCDE-pSB1C3<br>
 +
·      Gel electrophoresis of colony PCR product from 10/9 PcomCDE-E0240<br>
 +
·      Bacterial inoculation of checked products (for sequencing) and streak plates<br>
 +
·      Check colony fluorescene for whole construct (PcomX)<br>
 +
·      Gel extraction and purification of E0240 and I20260<br>
 +
·      Gel extraction and purification of PcomCDE-E0240<br>
 +
·      Streak plate for K880005-<i>comE</i>-C-myc<br>
 +
 
 +
·   <br>
 +
 
 +
<br><b>Sept 12</b> <br>
 +
Wet lab<br>
 +
·      Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br>
 +
·      Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Make glycerol stock for PcomCDE-pSB1C3<br>
 +
·      Gel electrophoresis of colony PCR product of PcomCDE-E0240 from 10/9; Bacterial inoculation of checked products (for sequencing); Streak plate<br>
 +
·      Check colony and fluorescence for whole construct (PcomX)<br>
 +
·      Streak plate for K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 15</b> <br>
 +
Wet lab<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      PCR check and inoculation of colonies of I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Restrict check, Gel extraction and Gel purification of pSB3K3 J04450 (CHL)<br>
 +
·   <br>
 +
 
 +
 
 +
<br><b>Sept 16</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 17</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
 +
·      Digestion check of  pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI<br>
 +
·      Digestion check of Combox + GFP on XmnI and PstI<br>
 +
·      Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at NotI and PstI<br>
 +
·      PCR check and inoculation for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Restrict check pSB3K3<br>
 +
·      Gel extraction and Gel purification of J04450 (CHL) insert X,S<br>
 +
·      Sequencing reaction for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 18</b> <br>
 +
Wet lab<br>
 +
·      Ligation of comX-myc into pSB1C3 cut on XbaI and PstI<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Colony PCR and inoculation of correct colonies for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Gel extraction and gel purification for J04450 (CHL), J04450 (KAN)<br>
 +
·      Sequencing reaction and Restrict check for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 19</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI<br>
 +
·      Colony PCR, inoculation for I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Miniprep for I20260/E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Gel extraction and gel purification J04450 (CHL), J04450 (KAN) XS<br>
 +
·      Sequencing reaction for K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 22</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Ligation of pSB3K3 backbone with 160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Gel extraction and gel purification of I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc EP<br>
 +
·      Colony PCR and inoculation of I20260/E0240-K880005-<i>comE</i>-C-myc <br>
 +
·      Gel extraction and gel purification J04450 (CHL), J04450 (KAN)<br>
 +
·      Oligo annealing and overnight ligation w/ pSB1C3for PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 23</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 of EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
 +
·      Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 ligated on Sept 22<br>
 +
·      Gel check and redo liagtion for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Gel extraction and purification for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Ethanol precipitation for sequencing, restrict check  K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Dephosphorylation and gel extraction and gel purification of J04450 (CHL) (X&S)<br>
 +
·      Gel extraction and Gel purification, dephosphorylation of J04450 (KAN) (E&P)<br>
 +
·      Oligo annealing, overnight ligation w/ pSB1C3 of PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 24</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification<br>
 +
·      Inoculation of bacterial colonies carrying pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Restrict check, gel extraction and purification, Ligation w/ pSB3K3 I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-·      myc, E0240-K880005-<i>comE</i>-C-myc <br>
 +
·      Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Transform pSB1C3-PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 25</b> <br>
 +
Wet lab<br>
 +
·      Restrcition digestion of ligated pSB3K3-160P<sub>combox</sub>-BBa_E0240<br>
 +
·      Restrict check, gel extraction and purification, Ligation w/ pSB3K3, I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Colony PCR w/ ‘NEW FPcomCDE’ and VR, inoculation of  pSB1C3-PcomCDE<br>
 +
 
 +
·   <br>
 +
 
 +
<br><b>Sept 26</b> <br>
 +
Wet lab<br>
 +
·      Transformation of pSB3K3-160P<sub>combox</sub>-BBa_E0240 sample1<br>
 +
·      Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240<br>
 +
·      Gel extraction and purificationI and ligation20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Inoculation for sequencing K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Transformation of pSB1C3-PcomCDE<br>
 +
 
 +
·   <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 29</b> <br>
 +
Wet lab<br>
 +
·   <br>
 +
 
 +
 
 +
<br><b>Sept 30</b> <br>
 +
Wet lab<br>
 +
·      Digestion of  RBS+comX-myc+B0015 on XbaI and PstI<br>
 +
·      Ligation of PcomCDE-E0240, both that day and overnight<br>
 +
·      Transformation<br>
 +
·   <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
</div>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<h3>Riboregulator</h3>
 +
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.7.1/jquery.min.js"></script>
 +
  <script>
 +
    $(document).ready(function(){
 +
      $('.head').each(function(){
 +
        var $content = $(this).closest('div').find('.content');
 +
        $(this).click(function(e){
 +
          e.preventDefault();
 +
          $content.not(':animated').slideToggle();
 +
    });
 +
});
 +
});
 +
  </script>
 +
  <style>
 +
    * { margin:0; padding:0; } /* a simple reset */
 +
 +
   
 +
 +
    .head {
 +
display:block;
 +
background:#FF9900;
 +
border-radius:2px;
 +
margin-bottom:5px;
 +
align:left;
 +
width:600px;
 +
height: 40px;
 +
z-index:-3;
 +
font-family:"Trebuchet MS", Helvetica, sans-serif;
 +
font-size: 35px;
 +
text-align: left;
 +
}
 +
 +
    .content {
 +
display:none;
 +
background:#8FF5D5;
 +
border-radius:10px;
 +
width:60%;
 +
margin-bottom:5px;
 +
align:center;
 +
z-index:-3;
 +
padding-left:100px;
 +
padding-right:100px;
 +
font-family:"Trebuchet MS", Helvetica, sans-serif
 +
}
 +
 +
   
 +
 
 +
  </style>
 +
 
 +
<div id="flight">
 +
<!--   J   U       U N       N EEEEEEEEEE   2 0 1 3---------------->
 +
<!--   J   U       U NN       N E   2 0 1 3---------------->
 +
<!--   J   U       U N N       N E   2 0 1 3---------------->
 +
<!--   J   U       U N  N   N E   2 0 1 3---------------->
 +
<!--   J   U       U N  N   N E   2 0 1 3---------------->
 +
<!--       J   U       U N    N   N EEEEEEEEEE    2 0 1 3---------------->
 +
<!--   J   U       U N    N   N E   2 0 1 3---------------->
 +
<!-- J   J   U       U N      N  N E       2 0 1 3---------------->
 +
<!-- J   J   U       U   N     N  N E   2 0 1 3---------------->
 +
<!-- J   J   U       U   N     N N E   2 0 1 3---------------->
 +
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU N       N EEEEEEEEEEE   2 0 1 3---------------->
 +
 
 +
 
 +
<!--   J   U       U L       Y            Y    2 0 1 3-------->
 +
<!--   J   U       U L           Y          Y 2 0 1 3-------->
 +
<!--   J   U       U L Y        Y 2 0 1 3-------->
 +
<!--   J   U       U L  Y      Y 2 0 1 3-------->
 +
<!--   J   U       U L                Y     Y        2 0 1 3-------->
 +
<!--       J   U       U L        Y  Y 2 0 1 3-------->
 +
<!--   J   U       U L            Y 2 0 1 3-------->
 +
<!-- J   J   U       U L    Y 2 0 1 3-------->
 +
<!-- J   J   U       U   L   Y 2 0 1 3-------->
 +
<!-- J   J   U       U   L   Y 2 0 1 3-------->
 +
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU LLLLLLLLLLLL     Y 2 0 1 3-------->
 +
 
 +
 
 +
 
 +
<div id="satu" align="center"> <h2><center>July 2014</center></h2>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 7 </b> <br>
 +
Wet lab<br>
 +
. Oligos phosphorylation <br>
 +
. ligation of TAs and CRs <br>
 +
. Colony PCR <br>
 +
. Digest Receptor Backbone pSB1C3 <br>
 +
. Agar-LB plates <br>
 +
. Inoculation for transformed Biobricks <br>
 +
. Primer Design for TA construct Gibson Assembly <br>
 +
. Gel check for the colony PCR <br>
 +
. P<sub>bad</sub> PCR amplification <br>
 +
 
 +
 
 +
<br> <b> July 8 </b> <br>
 +
Wet lab<br>
 +
. Transformation of ligated TAs and CRs <br>
 +
. Miniprep previous TAs CRs constructs <br>
 +
. restriction check previous TAs CRs constructs <br>
 +
. Colony PCR for CRs and TAs <br>
 +
. Re-ligation of TAs and CRs <br>
 +
. P<sub>bad</sub> PCR clean up and Digestion <br>
 +
. Ligation of P<sub>bad</sub> with pSB1C3 <br>
 +
. Sequencing preparation of P<sub>bad</sub> <br>
 +
 
<br> <b> July 9</b> <br>
<br> <b> July 9</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Extraction of BBa_J176171, BBa_K817002 P<i>fadBA</i> and pEGFP-N1 by miniprep<br>
+
. Transformation of Re-ligated TAs and CRs <br>
-
·      Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification<br>
+
. Colony PCR <br>
-
·      Restriction of BBa_J176171 for P<i>fadBA</i> Ase1 and BamH1; FABP1 promoter by Xba1 and Not1<br>
+
. Gibson assembly master mix preparation <br>
-
·      Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br>
+
-
·      PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
-
·      <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> (<i>fad</i>R) transformation<br>
+
<br> <b> July 10</b> <br>
<br> <b> July 10</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter<br>
+
. Sequencing preparation of P<sub>bad</sub> <br>
-
·      PCR for FABP1 promoter, PCR replication<br>
+
. Miniprep previous TAs CRs constructs <br>
-
·      BBa_J176171 vector dephosphorylation  by antartic phosphatase<br>
+
. restriction check previous TAs CRs constructs <br>
-
·      Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br>
+
. Colony PCR <br>
-
·      BBa_J52034 (<i>fad</i>R) inoculation<br>
+
. Inoculation for glycerol stocks <br>
-
·      Digestion for BBa_K817002 P<i>fadBA</i> extraction<br>
+
. Digest Receptor Backbone , digestion of BBa_ BBa_E0240 with EcoR1-HF and Xbal and gel purification <br>
-
Gel check for BBa_K817002 P<i>fadBA</i> extraction<br>
+
<br> <b> July 11</b> <br>
<br> <b> July 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Gel purification for BBa_K817002 P<i>fadBA</i> extraction<br>
+
. P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <br>
-
·      Gel check for FABP1 promoter PCR product<br>
+
. Ligation of P<sub>bad</sub> and BBa_E0240 <br>
-
·      PCR clean-up<br>
+
. Overlapping PCR design <br>
-
·      BBa_J52034 (<i>fad</i>R) miniprep<br>
+
. Gibson assembly master mix preparation <br>
-
·      BBa_J52034 restriction by Pst1 HF and Not1 HF<br>
+
. Glycerol stock <br>
-
·      PCR  for PPAR-alpha promoter<br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
-
Dry lab<br>
+
. Run gel for yesterday's restriction check <br>
-
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions<br>
+
. Miniprep BBa_E0240, mRFP, BBa_B0015 <br>
 +
. Colony PCR <br>
 +
. One full set re-ligation (short time) <br>
 +
. Miniprep BBa_I13401 and BBa_K592009 <br>
-
<br> <b> July 12</b> <br>
 
-
Wet lab<br>
 
-
·  Digestion of FABP1 promoter PCR product<br>
 
-
·      Ran gel for PCR check of PPAR-alpha promoter<br>
 
-
·      Digest pEGFP-n1 for BBa_J52034 <i>fad</i>R<br>
 
-
·      Ran gel for pEGFP-n1 for BBa_J52034 <i>fad</i>R followed by gel purification<br>
 
-
·      BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and P<i>fadBA</i><br>
 
-
Dry Lab<br>
 
-
Primers design for pCMV cloning for <i>fad</i>R expression<br><br>
 
       </div>
       </div>
   </div>
   </div>
   <div>
   <div>
-
       <a href="#" class="head">Week 3</a>
+
       <a href="#" class="head">Week 2</a>
-
       <div class="content" align="left">
+
       <div class="content" align="left" style="display: none;">
-
<br><b>July 15</b> <br>
+
<br> <b> July 14 </b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation<br>
+
. Gibson assembly master mix preparation <br>
-
·      Transformation of  FABP1 promoter, EGFP, and BBa_J176171 ligation<br>
+
. PCR <br>
-
·      FABP1 promoter+EGFP +BBa_J176171 ligation restriction check<br>
+
. Ligation of P<sub>bad</sub> an BBa_E0240 <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI) <br>
 +
. One full set of re-ligation (short time) <br>
 +
. One full set of re-ligation (O/N) <br>
 +
. Preparations for P<sub>bad</sub> re-Characterization <br>
-
<br><b>July 16</b> <br>
 
-
Wet Lab<br>
 
-
·      Miniprep for full construct of FABP1 promoter and pEGFP-N1<br>
 
-
·      <i>fad</i>R and pEGFP-N1 Backbone parts ligation <br>
 
-
·      Plasmids extraction for pCMV cloning for <i>fad</i>R<br>
 
-
·      BBa_K817002 P<i>fadBA</i> promoter extraction by EcoR1 and Pst1 HF<br>
 
-
·      BBa_J52034 restriction by EcoR1 and Pst1 HF<br>
 
-
·      Inoculations for full construct of FABP1 promoter and pEGFP-N1<br>
 
-
·      Streak colonies containing the right construct for FABP1 promoter<br>
 
-
<br><b>July 17</b> <br>
+
<br> <b> July 15 </b> <br>
Wet lab<br>
Wet lab<br>
-
·      Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep<br>
+
. Inoculation for P<sub>bad</sub> -GFP-TT <br>
-
·      Repeat <i>fad</i>R and P<i>fadBA</i> constructs<br>
+
. (2nd time) Colony PCR using VF2 and VR <br>
-
·      Digest pEGFP-n1 and BBa-J176171 for <i>fad</i>R<br>
+
. Design primer for P<sub>bad</sub> promoter sequencing <br>
-
·      Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br>
+
. Gibson assembly master mix preparation <br>
-
·      Gel check and gel extraction for BBa_J176171 for <i>fad</i>R and BBa_K817002 (P<i>fadBA</i>) <br>
+
. Preparations for P<sub>bad</sub> re-characterization <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. One full set re-ligation (O/N) <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
 
-
<br><b>July 18</b> <br>
+
<br> <b> July 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Gel purification for all digested products<br>
+
. Gibson assembly master mix preparation <br>
-
·      FABP1 promoter digestion check for whole construct prior transfection<br>
+
. Preparations for P<sub>bad</sub> re-characterization <br>
-
·      P<i>fadBA</i> vector de phosphorylation and ligation with EGFP and BBa_J176171<br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
-
·      Transformation of P<i>fadBA</i>+EGFP+BBa_J176171<br>
+
. Prepare receptor backbone for oligo anealing <br>
 +
. inoculate successful oligo annealing colonies <br>
 +
. Oligo annealing products C. PCR? <br>
-
<br><b>July 19</b> <br>
+
<br> <b> July 17</b> <br>
Wet lab<br>
Wet lab<br>
-
·      FABP1 promoter preparation for transfection<br>
+
. Gibson assembly master mix preparation <br>
-
·      Ligation of P<i>fadBA</i> and BBa_J176171 followed by transformation<br>
+
. P<sub>bad</sub> re-characterization <br>
-
·      PCR for PPAR-alpha promoter cloning<br>
+
. Miniprep CRs TAs <br>
-
Dry lab<br>
+
. Restriction check CRs,TAs <br>
-
·      Design for characterization of the transfected cells with FABP1 construct<br>
+
. Oligo annealing products C. PCR <br>
-
·      Review for Multiple Sites Mutagenesis<br>
+
. Prepare receptor backbone for oligo anealing <br>
-
·      Ordered primers for pCMV cloning from pEGFP-N1<br><br>
+
. Restrict CRs plasmids for GFP.dTT <br>
 +
. Restrict TAs plasmids for dTT <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict dTT for TAs <br>
 +
. Ligate CR.GFP.dTT <br>
 +
. Ligate TA.dTT <br>
 +
. Transform Ligated CR.GFP.dTT <br>
 +
. Transform Ligated TA.dTT <br>
 +
. Transform-Ligate Annealed oligos <br>
 +
 
 +
<br> <b> July 18</b> <br>
 +
Wet lab<br>
 +
. Gibson assembly for amilCP <br>
 +
. P<sub>bad</sub>  re-characterization <br>
 +
. Colony PCR <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict dTT for TAs <br>
 +
. M9 mininaml medium preparation <br>
 +
. Colony PCR <br>
 +
. plate streaking for P<sub>bad</sub>-BBa_E0240 <br>
 +
. inoculation of TAs and CRs <br>
 +
 
 +
<br> <b> July 19</b> <br>
 +
Wet lab<br>
 +
. P<sub>bad</sub> re-characterization <br>
 +
 
       </div>
       </div>
Line 390: Line 1,458:
   <div>
   <div>
-
       <a href="#" class="head">Week 4</a>
+
       <a href="#" class="head">Week 3</a>
-
       <div class="content" align="left">
+
       <div class="content" align="left" style="display: none;">
-
<br><b>July 22</b> <br>
+
<br> <b> July 21 </b> <br>
Wet lab<br>
Wet lab<br>
-
·  FABP1 construct given for characterization and transfection<br>
+
. Gibson assembly for amilCP <br>
-
·  Further colony screening for FABP1 construct, inoculations<br>
+
. Miniprep CRs TAs <br>
-
·  Inoculation of P<i>fadBA</i> and BBa_J176171<br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
-
·  Ran gel for PPAR-alpha promoter PCR products<br>
+
. Restrict GFP.dTT for CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict BBa_B0015 EcoR1 and XbaI <br>
 +
. BBa_J04450 backbone extraction <br>
 +
. Colony PCR <br>
 +
. inoculation of TAs and CRs <br>
 +
. prepare P<sub>bad</sub> L(+) A. concentration gradients for characterization <br>
 +
. Restrict CRs <br>
 +
. Restrict TAs-Version1 <br>
 +
. Restrict TAs-Version2 <br>
 +
. Ligate CR.GFP.dTT <br>
 +
. Ligate TA.dTT <br>
 +
. Transform P<sub>bad</sub>.TA <br>
 +
. Transform CR.GFP.dTT <br>
 +
. Transform Gibson assembly <br>
 +
 
-
<br><b>July 23</b> <br>
+
<br> <b> July 22 </b> <br>
Wet lab<br>
Wet lab<br>
-
·  Primers arrival for pCMV cloning<br>
+
. Colony PCR <br>
-
·  PCR for pCMV cloning<br>
+
. Miniprep CRs TAs <br>
-
·  Digestion and gel for construction check for FABP1 promoter<br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
-
·  P<i>fadBA</i> gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation<br>
+
. Restrict BBa_B0015-Version2 <br>
-
·  Digestion of pEGFP-N1 for <i>fad</i>R, followed by gel check and extraction<br>
+
-
<br><b>July 24</b> <br>
+
 
 +
<br> <b> July 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      DNA purification from gel extraction for digested pEGFP-N1<br>
+
. Inoculation candidate colony or construct <br>
-
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
+
. Miniprep and restriction check inoculations <br>
-
·      Ran gel for pCMV cloning from pEGFP-N1<br>
+
. Gibson assembly check <br>
-
·      PCR clean up for pCMV cloning from pEGFP-N1<br>
+
. Gibson PCR <br>
-
·      PCR for PPAR-alpha promoter with new primers using Taq polymerase<br>
+
. Teach inoue competent cells to main project <br>
-
·      Transformation of mutagenesis products<br>
+
. Miniprep of BBa_B0015, P<sub>bad</sub>-BBa_E0240 <br>
-
·      Inoculation of P<i>fadBA</i> ligation colonies<br>
+
. glycerol stock (P<sub>bad</sub>-BBa_E0240) <br>
 +
. Re-ligation of oligo annealing O/N <br>
-
<br><b>July 25</b> <br>
+
<br> <b> July 24</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br>
+
. miniprep candidate colony or construct <br>
-
·      Minprep of P<i>fadBA</i>+EGFP+BBa_J176171 ligation colonies, followed by digestion check <br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
 +
. Re-Re-ligation transformation <br>
 +
. Re-Re-ligation transformation V.X <br>
 +
. Gibson assembly check <br>
-
<br><b>July 26</b> <br>
+
<br> <b> July 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
+
. Certification day (safety evaluation) <br>
-
·      Ran gel before parental string digestion of mutagenesis products<br>
+
-
·      Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check<br>
+
-
·      Plasmids preparation according to transfection requirements, construct and controls<br><br>
+
       </div>
       </div>
Line 433: Line 1,517:
   <div>
   <div>
-
       <a href="#" class="head">Week 5</a>
+
       <a href="#" class="head">Week 4</a>
-
       <div class="content" align="left">
+
       <div class="content" align="left" style="display: none;">
-
<br><b>July 30</b> <br>
+
<br> <b> July 28 </b> <br>
Wet lab<br>
Wet lab<br>
-
·      PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
+
. oligo phosphorylation <br>
-
·      Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br>
+
. oligo annealing <br>
-
·      BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b<br>
+
. ligation of annealed oligos O/N <br>
-
·      PCR for PPAR-alpha promoter with new primers using Vent polymerase<br>
+
. gel for C. PCR <br>
-
Dry lab<br>
+
. receptor backbone for annealed oligos <br>
-
·      Discussion and re assessment of constructs related to pEGFP-N1<br>
+
. inoculate last ligation <br>
 +
. Check past locks/keys status <br>
 +
. P<sub>bad</sub> sequencing <br>
 +
. transformation of some of the existing riboregulator Biobricks <br>
 +
. Benchling Check <br>
-
<br><b>July 31</b> <br>
+
<br> <b> July 29 </b> <br>
-
·      Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
+
Wet lab<br>
-
·      PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up<br>
+
. Miniprep and restriction check inoculations <br>
-
·      PCR for PPAR-alpha promoter with reference primers using Vent polymerase<br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
-
·      Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase<br><br>
+
. Inoculation of CR and TA from distribution kit <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
. prepare Side project Buffer <br>
 +
. prepare side project gel red 200:1 <br>
 +
. Check digested GFP <br>
 +
. Check digested BBa_B0015 <br>
 +
. ligate oligos into GFP <br>
 +
. ligate oligos into BBa_B0015 <br>
 +
. prepare M9 for characterization <br>
 +
. transform our P<sub>bad</sub> into DH5alpha <br>
 +
. prepare M9 medium <br>
 +
. transformation of Clon001-010 in pSB1C3 ( with a control) <br>
 +
 
 +
 
 +
<br> <b> July 30</b> <br>
 +
Wet lab<br>
 +
. transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401) <br>
 +
. C. PCR for yesterday transformations (CLON001-010) <br>
 +
. If construct after gel check inoculation, restriction, ligation <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
. transform our P<sub>bad</sub>into DH5alpha <br>
 +
. prepare M9 for characterization <br>
 +
. Teach inoue competent cells to main project <br>
 +
 
 +
<br> <b> July 31</b> <br>
 +
Wet lab<br>
 +
. Try Gibson assembly mastermix <br>
 +
. Digest Biobrick TA and CR <br>
 +
. PCR clean up for existing CR and TA Biobricks <br>
 +
. run gel for yesterday's colony PCR <br>
 +
. check dNTPs <br>
 +
. Sequencing preparation for possible construct <br>
 +
. colony PCR for 5th <br>
 +
. inoculate candidates <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
 
       </div>
       </div>
   </div>
   </div>
  </div>
  </div>
 +
<!------      AAAAAAAAAAAA    U        U  GGGGGGGGGGGG    U          U    SSSSSSSSSSSSS    TTTTTTTTTTTTTT      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                      T      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                  T      2 0 1 3---------------->
 +
<!------      A      A   U        U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      A      A   U     U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      AAAAAAAAAAAA   U        U  G              U          U    SSSSSSSSSSSSS          T        2 0 1 3---------------->
 +
<!------      A        A   U        U  G    GGGGGGG    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T 2 0 1 3---------------->
 +
<!------      A        A   U        U G          G    U          U                S        T 2 0 1 3---------------->
 +
<!------      A        A   UUUUUUUUUUU  GGGGGGGGGGGG    UUUUUUUUUUUUU    SSSSSSSSSSSSS        T        2 0 1 3---------------->
-
<div id="satu" align="center"> <h1>August 2013</h1>
+
 
 +
<div id="satu" align="center"> <h2>August 2014</h2>
    
    
<div>
<div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
-
       <div class="content" align="left">
+
       <div class="content" align="left" style="display: none;">
-
<br><b>August 2</b> <br>
+
<br><b>Aug 1</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. colony PCR for 5th <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Digest Biobrick TA and CR <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. short ligation for TAs CRs <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Transform TAs, CRs <br>
 +
. inoue Buffer <br>
 +
. inoculations if possible <br>
 +
. miniprep CLON005 <br>
 +
. Digest 005 Spe1 and Pst1 <br>
 +
. ligate CLON 005 with BBa_B0015 <br>
 +
. prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// BBa_J23102 X, P <br>
 +
. Ligate P<sub>bad</sub> with Key1 dTT <br>
 +
. digest P<sub>tet</sub> lock 1 with S and P <br>
 +
 
 +
<br><b>Aug 2</b> <br>
 +
Wet lab<br>
 +
. Devise a general blue print for the catalog and feature page <br>
 +
 
       </div>
       </div>
     </div>
     </div>
 +
<div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 4</b> <br>
 +
Wet lab<br>
 +
. Mini prep 001-010 <br>
 +
. Restrict TAs <br>
 +
. restrict CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict BBa_B0015 <br>
 +
. Ligate P<sub>bad</sub>.TA <br>
 +
. ligate CR.GFP.dTT <br>
 +
. Ligate P<sub>bad</sub>.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. inoculate 001-010 <br>
 +
. Sequence 001-010 <br>
 +
. inoculate BBa_B0015 <br>
 +
. inoculate constitutive promoter <br>
 +
. inoculate P<sub>bad</sub> with key 1 dTT <br>
 +
. Competent cells preparations DH10B <br>
 +
. Prepare for functional assay of P<sub>bad</sub> promoter <br>
 +
. Prepare M9 minimal Medium <br>
 +
. make a second list of oligos to be sent out <br>
 +
. Test out GA mastermix efficiency <br>
 +
. Ligate TAs oligo with BBa_B0015 <br>
 +
. inoue Buffer <br>
 +
. Sequence 001-010 <br>
 +
<br><b>Aug 5</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for TA.dTTs <br>
 +
. inoculate TA.dTT <br>
 +
. ligation of P<sub>bad</sub> and key3 <br>
 +
. Restrict TAs <br>
 +
. restrict CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Ligate P<sub>bad</sub>.TA <br>
 +
. ligate CR.GFP.dTT <br>
 +
. Ligate P<sub>bad</sub>.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. Prepare M9 medium <br>
 +
. Prepare inoculations for P<sub>bad</sub> characterization <br>
 +
. Sequecing preparation <br>
 +
. Digest P<sub>tet</sub> lock 1 with S and P <br>
 +
. Glycerol stock for 001-010 <br>
 +
. Inoue competent cells <br>
 +
. "Mini Prep pSB1C3-PCR P<sub>bad</sub><br>
 +
<br><b>Aug 6</b> <br>
 +
Wet lab<br>
 +
. Miniprep TA.dTT <br>
 +
. Restriction check for TA.dTT <br>
 +
. Miniprep TAs CRs <br>
 +
. Restrict TAs <br>
 +
. Restrict CRs <br>
 +
. Restrict BBa_B0015 for TAs <br>
 +
. Colony PCR for TA.dTTs <br>
 +
. Oligo annealing for TA.dTT <br>
 +
. Inoue competent cells <br>
 +
. Sequencing results <br>
 +
. Ligate P<sub>bad</sub> with Key1 dTT <br>
 +
. Ligate P<sub>tet</sub> Lock 1 (GFP dTT) <br>
 +
. Send PCR P<sub>bad</sub> for sequencing <br>
 +
. Assay P<sub>bad</sub> promoter <br>
 +
<br><b>Aug 7</b> <br>
 +
Wet lab<br>
 +
. Gel check for C. PCR <br>
 +
. inoculate possible TA.dTT <br>
 +
. Ligation of TA and dTT (4TAs) <br>
 +
. Ligation of P<sub>bad</sub> and key dTT <br>
 +
. Gel check for TAs CRs <br>
 +
. competent cells <br>
 +
. miniprep P<sub>bad</sub>-dTT <br>
 +
. miniprep P<sub>bad</sub>-key3 <br>
 +
. Colony PCR for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
 +
. Restriction of P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
 +
. prepare backbone and or insert if any // and restriction check <br>
 +
. Preparation for P<sub>bad</sub> promoter characterization <br>
 +
. Colony PCR for P<sub>bad</sub> with Key 1 dTT <br>
 +
. Colony PCR for P<sub>tet</sub> Lock 1 GFP dTT <br>
 +
. inoculation of ligated DNA <br>
 +
<br><b>Aug 8</b> <br>
 +
Wet lab<br>
 +
. digestion of P<sub>bad</sub>-key3 <br>
 +
. Restriction check for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3-DTT <br>
 +
. ligation of P<sub>bad</sub>-key3-dTT <br>
 +
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. Miniprep P<sub>bad</sub> Key 1 dTT <br>
 +
. Miniprep P<sub>tet</sub> Lock 1 GFP dTT <br>
 +
. Restriction Check for the key 1 and lock 1 mentioned above <br>
 +
. Prepare digestion of key 1 and lock 1 for ligation <br>
 +
. Transform BBa_J13002 <br>
 +
. Restriction check CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. digestion of CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. ligation of CLON007(J), CLON 010(I) and CLON (G) with P<sub>bad</sub> <br>
 +
. C. PCR for CLON 022-025 TAs.dTT <br>
 +
. ligation of P<sub>bad</sub> and TAs <br>
 +
. ligation of GFP and CRs <br>
 +
. Inoue competent cells <br>
 +
. P<sub>bad</sub> promoter characterization <br>
 +
<br><b>Aug 9</b> <br>
 +
Wet lab<br>
 +
. transformation , all in CHL, overnight ligation 16C <br>
 +
. Colony PCR gel check <br>
 +
. Check competent cells <br>
-
+
<br><b>Aug 10</b> <br>
-
<hr>
+
Wet lab<br>
-
+
. Restriction check for TAs.dTT <br>
-
<table class= "site_map_table" align= "center">
+
. Gel check for C. PCR TAs.dTT <br>
-
<tr>
+
-
<td class= 'site_map_column'><a href="#"><h4>Home</h4></a>
+
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Team</li></a>
+
-
<li class='site_link'><a href="#">Project</li></a>
+
-
<li class='site_link'><a href="#">WetLab</li></a>
+
-
<li class='site_link'><a href="#">Achievement</li></a>
+
-
<li class='site_link'><a href="#">Human Practice</li></a>
+
-
<li class='site_link'><a href="#">Side Project</li></a>
+
-
</ul>
+
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Team</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Member</li></a>
 
-
<li class='site_link'><a href="#">Advisers</li></a>
 
-
<li class='site_link'><a href="#">Instructor</li></a>
 
-
<li class='site_link'><a href="#">Attribution</li></a>
 
-
<li class='site_link'><a href="#">Acknowledgement</li></a>
 
-
</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Main Project</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Overview</li></a>
 
-
<li class='site_link'><a href="#">Module one</li></a>
 
-
<li class='site_link'><a href="#">Module two</li></a>
 
-
<li class='site_link'><a href="#">Data</li></a>
 
-
<li class='site_link'><a href="#">Characterisation</li></a>
 
-
</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Side Project</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Overview</li></a>
 
-
<li class='site_link'><a href="#">Parts</li></a>
 
-
<li class='site_link'><a href="#">Catalogue</li></a>
 
-
<li class='site_link'><a href="#">Feature Pages</li></a>
 
-
</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Human Practice</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Overview</li></a>
 
-
<li class='site_link'><a href="#">Start up kit</li></a>
 
-
<ul>
 
-
<li class='site_link'><a href="#">Interview</li></a>
 
-
<li class='site_link'><a href="#">Report</li></a>
 
-
<li class='site_link'><a href="#">Database</li></a>
 
-
</ul>
 
-
<li class='site_link'><a href="#">Talks</li></a>
 
-
<li class='site_link'><a href="#">Outreach</li></a>
 
-
</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Wetlab</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Protocol</li></a>
 
-
<li class='site_link'><a href="#">Notebook</li></a>
 
-
<li class='site_link'><a href="#">Safety</li></a>
 
-
</ul>
 
-
</td>
 
-
<td class= 'site_map_column'><a href="#"><h4>Achievement</h4></a>
 
-
<ul class= 'site_list'>
 
-
<li class='site_link'><a href="#">Medal Requirements</li></a>
 
-
<li class='site_link'><a href="#">Deliverable</li></a>
 
-
</ul>
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
+
      </div>
-
+
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 11</b> <br>
 +
Wet lab<br>
 +
. Restriction check for TAs.dTT <br>
 +
. Gel check for C. PCR TAs.dTT <br>
 +
. Colony PCR for CR.GFP.dTTs <br>
 +
. Colony PCR for P<sub>bad</sub>.TAs <br>
 +
. Inoculate CR.GFP.dTTs <br>
 +
. Inoculate P<sub>bad</sub>.TAs <br>
 +
. Restrict TA.dTTs <br>
 +
. Ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. Transform P<sub>bad</sub>.TA.dTTs <br>
 +
. Restriction check for P<sub>tet</sub> Lock 1 GFP dTT and P<sub>bad</sub> Key 1 dTT <br>
 +
. Transform 2014 Kit plate 4 well GF <br>
 +
. Transformation of restricted checked DNA <br>
 +
. inoculate BBa_J13002 <br>
 +
. inoculate more P<sub>bad</sub> dTTs <br>
 +
. sequencing PCR for MCR, MTA <br>
 +
. transformation for backbone with KAN resistant <br>
 +
. inoculation B0034 from glycerol stock B0034 <br>
 +
. restriction check <br>
 +
. Ligation of P<sub>bad</sub>-key3 into BBa_B0015-pSB1AK3 plasmid <br>
 +
. inoculation of P<sub>bad</sub>-key3 <br>
 +
 
 +
<br><b>Aug 12</b> <br>
 +
Wet lab<br>
 +
. transformation P<sub>bad</sub> -TA <br>
 +
. Gel for TA.dTTs <br>
 +
. GEl for C. PCR for CR.GFP.dTT <br>
 +
. Digestion of PCR P<sub>bad</sub> <br>
 +
 
 +
<br><b>Aug 13</b> <br>
 +
Wet lab<br>
 +
. miniprep CR.GFP.dTTs <br>
 +
. miniprep P<sub>bad</sub> <br>
 +
. restriction check CR.GFP.dTTs <br>
 +
. inoculate TA.dTTs <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. transform, P<sub>bad</sub>.Key3 <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
. Gibson Assembly mastermix <br>
 +
. Digest P<sub>bad</sub> Key dTT and P<sub>tet</sub> Lock 1 GFP dTT for 3A assembly <br>
 +
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. 3A assembly <br>
 +
. Plan for P<sub>bad</sub> characterization <br>
 +
. Prepare stock arabinose solution M9 medium <br>
 +
 
 +
<br><b>Aug 14</b> <br>
 +
Wet lab<br>
 +
. Gel check for lock3.GFP.dTT colony PCR I-VI <br>
 +
. check dNTPs <br>
 +
. miniprep TA.dTTs <br>
 +
. restriction check TA.dTTs <br>
 +
. miniprep Lock3.GFP.dTT <br>
 +
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. ligate TA.dTT with P<sub>bad</sub> <br>
 +
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
 
 +
<br><b>Aug 15</b> <br>
 +
Wet lab<br>
 +
. Gibson Assembly mastermix <br>
 +
. purify CR.GFP.dTTs <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. Digestion of TA-dTT and PCR clean-up <br>
 +
. Restriction Check for TA-dTT <br>
 +
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
. make 10x DNA loading Dye <br>
 +
. Order new TA and CR <br>
 +
. Gibson Assembly mastermix <br>
 +
 
 +
 
 +
<br><b>Aug 16</b> <br>
 +
Wet lab<br>
 +
. Miniprep <br>
 +
. Colony PCR for final constructs <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 18</b> <br>
 +
Wet lab<br>
 +
. Restriction check for P<sub>bad</sub>.TA.dTTs <br>
 +
. Restriction check for constitutive promoter.CR.GFP.dTT <br>
 +
. Restriction check for constitutive promoter.RBS.GFP.dTT <br>
 +
. PCR for CR.GFP.dTT <br>
 +
. PCR for RBS.GFP.dTT <br>
 +
. PCR check for constitutive promoter.CR.GFP.dTT <br>
 +
. PCR check for constitutive promoter.RBS.GFP.dTT <br>
 +
. PCR check for P<sub>bad</sub>.TA.dTT <br>
 +
. restriction check TA.dTTs <br>
 +
. Prepare functional assay for RBS, CRs <br>
 +
. Inoculate candidates <br>
 +
. Prepare pSB3K3 <br>
 +
. Prepare pSB1C3 for oligo ligation <br>
 +
. Prepare BBa_I13401 for oligo ligation <br>
 +
. Prepare BBa_B0015 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. Prepare for P<sub>bad</sub> characterization <br>
 +
. Take over the tank B and C around 1pm-5;30 <br>
 +
 
 +
<br><b>Aug 19</b> <br>
 +
Wet lab<br>
 +
. Gel check for O/NPCR products <br>
 +
. Gel check for PCR products (18/08) <br>
 +
. miniprep c.Promoter.Lock 1RBS.GFP.dTT <br>
 +
. miniprep possible candidates <br>
 +
. Restrict TA.dTT <br>
 +
. pSB1C3-BBa_I13401 for oligo ligation <br>
 +
. BBa_B0015 for oligo ligation <br>
 +
. pSB1C3-BBa_J04450 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. P<sub>bad</sub>-key3-dTT restriction check <br>
 +
 
 +
<br><b>Aug 20</b> <br>
 +
Wet lab<br>
 +
. Gel check for P<sub>bad</sub>.TA.dTTs <br>
 +
. purification for BBa_B0015, BBa_I13401 <br>
 +
. pSB1C3-BBa_J04450 for oligo ligation <br>
 +
. Gel check for PCR products (19/08) <br>
 +
. PCR check for BBa_J23102, LOCK 3 GFP DTT <br>
 +
. Restriction check for key1 DTT <br>
 +
 
 +
<br><b>Aug 21</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for final constructs <br>
 +
. Ligate UST lock <br>
 +
. restriction check for key1.dTT <br>
 +
. ligate P<sub>bad</sub>.key1.dTT <br>
 +
. ligate lock 3.GFP.dTT with C. promoter <br>
 +
. Colony PCR for c. promoter.MCR.GFP.dTT <br>
 +
. competent cells <br>
 +
. Glycerol stocks <br>
 +
. transform HKUSTlock.GFP.dTT <br>
 +
. Transform pSB1C3-HKUSTlock <br>
 +
 
 +
 
 +
<br><b>Aug 22</b> <br>
 +
Wet lab<br>
 +
. competent cells <br>
 +
. Colony PCR for pSB1C3-HKUSTlock1 <br>
 +
. Colony PCR for HKUSTlock1.GFP.dTT <br>
 +
. miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. restrict pSB3K3 for 3 pieces assemblage <br>
 +
. ligate TA-CR into pSB3K3 <br>
 +
. Transform TA-CR <br>
 +
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
 +
. inoculate P<sub>bad</sub>.key1.dTT <br>
 +
. gel check for C. PCR on 21/08 <br>
 +
. extraction of pSB3K3 <br>
 +
. inoculate HKUSTlock1.GFP.dTT <br>
 +
. inoculate pSB1C3-HKUSTlock1 <br>
 +
 
 +
 
 +
<br><b>Aug 23</b> <br>
 +
Wet lab<br>
 +
. Gel check for TAs <br>
 +
. colony PCR for UST lock in BBa_I13401 and pSB1C3 <br>
 +
. transform ust key <br>
 +
. Check competent cells <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 24</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
 +
. Gel check for P<sub>bad</sub>.Key1.dTT <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 25</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
 +
. PCR check for CR.GFP.dTT <br>
 +
. PCR check for Cons. prom.CR.GFP.dTT <br>
 +
. anneal and ligate ust lock <br>
 +
. anneal and ligate ust key <br>
 +
. transform ust lock1 <br>
 +
. transform ust key <br>
 +
. ligation again key 1 into BBa_B0015 <br>
 +
 
 +
 
 +
<br><b>Aug 26</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for "un-named lock" <br>
 +
. inoculate ones which SEEMS like it contains lock <br>
 +
. Colony PCR for "un-named key" <br>
 +
. inoculate ones which SEEMS like it contains key <br>
 +
. colony PCR for Key1.DTT <br>
 +
. Gel check for O/NPCR products <br>
 +
. PCR check for CR.GFP.dTTs <br>
 +
. Inoculate all possible candidates <br>
 +
. Prepare BBa_I13401 for CRs <br>
 +
. Streak possible c.p.CR/RBS.GFP.dTT <br>
 +
. make SOC <br>
 +
. single cut check for TAs <br>
 +
. make 5 alpha cell <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 27</b> <br>
 +
Wet lab<br>
 +
. miniprerp candidates <br>
 +
. PCR check for candidates <br>
 +
. gel check for o/n PCR <br>
 +
. restriction check for ust key 1 <br>
 +
. restriction check for key 1 <br>
 +
. P<sub>bad</sub> for ust key 1 <br>
 +
. extract ust lock 1 for BBa_J23102 <br>
 +
. ligate ust lock and key <br>
 +
. transform ust lock and key <br>
 +
. prepare plates for macroscope <br>
 +
. prepare dna for sequencing <br>
 +
. SOB <br>
 +
. SOC <br>
 +
. oligo annealing <br>
 +
. plasmid PCR for HKUSt lock and key <br>
 +
. overnight digestion of B0034 for control construct <br>
 +
. Digestion of HKUST key-BBa_B0015 <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 28</b> <br>
 +
Wet lab<br>
 +
. miniprep locks <br>
 +
. glycerol stocks for locks <br>
 +
. minipreps for BBa_B0015 and B0034 <br>
 +
. ligation of P<sub>bad</sub>-Hkust key-BBa_B0015 <br>
 +
. digestion of HKUST lock <br>
 +
. ligation of HKUST lock with GFP <br>
 +
. ligation of HKUST key and lock (4th) <br>
 +
. ligation of B0034 with GFP <br>
 +
. colony PCR of HKUST key and lock <br>
 +
. inoculation of key and lock <br>
 +
. miniprep <br>
 +
. Key one restriction check <br>
 +
. key one restriction for inserting P<sub>bad</sub> <br>
 +
. Restrict and extract lock3c rbs and ligate into c.promoter <br>
 +
. ligate lock 3c.GFP.dTT with C. promoter <br>
 +
. transform c. promoter lock 3c rbs.GFP.DTT <br>
 +
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into pSB3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. BBa_E0240 ligate with C. Promoter <br>
 +
. TB <br>
 +
. parallel ligation and anealing for key 1 <br>
 +
. Sequencing preparations <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 29</b> <br>
 +
Wet lab<br>
 +
. minipreps <br>
 +
. Gel extraction, purification for O/N digestions <br>
 +
. competent cells <br>
 +
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into pSB3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. PCR check for Biobrick key1 <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 30</b> <br>
 +
Wet lab<br>
 +
. inoculation of 12 falcons <br>
 +
. Colony PCR for pSB3K3 constructs <br>
 +
. Transform key1 O/N <br>
 +
. miniprep <br>
 +
. Competent cells <br>
 +
. AseI check Key 1 Dtt <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
  </div>
 +
 
 +
<!---- SSSSSSSS  EEEEEEEE  PPPPPPPP  TTTTTTTTTTTT  EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  RRRRRRRR    2 0 1 3-------->
 +
<!---- S          E          P      P      T        E          MM      MM  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M M    M M  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M  M  M  M  B      B  E          R  R 2 0 1 3-------->
 +
<!----     S          E          P      P        T        E          M  M M  M  B      B  E          R    R 2 0 1 3-------->
 +
<!----      SSSSSSSS  EEEEEEEE  PPPPPPPP        T        EEEEEEEE  M    M    M  BBBBBBBB    EEEEEEEEE  RRRRRRRR 2 0 1 3-------->
 +
<!----            S  E          P              T        E          M        M  B      B  E          R  R      2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R  2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R 2 0 1 3-------->
 +
<!----   S  E          p              T        E          M        M  B      B  E          R      R 2 0 1 3-------->
 +
<!---- SSSSSSSS  EEEEEEEE  P              T        EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  R  R 2 0 1 3-------->
 +
 
 +
<div id="satu" align="center"> <h2>September 2014</h2>
 +
 
 +
<div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 1</b> <br>
 +
Wet lab<br>
 +
. restriction check P<sub>bad</sub>- Lost your key-DTT <br>
 +
. restriction check P<sub>bad</sub>-Lost your key-DTT <br>
 +
. colony PCR for CR.TAs (orthogonality) <br>
 +
. colony PCR for Key1,DTT <br>
 +
. ligate lock3c.GFP.dTT into c.p plasmid <br>
 +
. transform lock3.GFP.DTT <br>
 +
. Colony PCR for CR.TAs (orthogonality) <br>
 +
. Colony PCR for sherLock-GFP-DTT <br>
 +
. restriction check for Lost Your Key in pSB1C3 <br>
 +
. transform P<sub>bad</sub>.lost your key.DTT <br>
 +
. inoculate orthogonality candidates <br>
 +
. Prepare midi-prep for pSB3K3 <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 2</b> <br>
 +
Wet lab<br>
 +
. Gel check for O/N colony PCR for key1 and pSB3K3 <br>
 +
. massive prep <br>
 +
. PCR check sense reverse <br>
 +
. colony PCR for orthogonality <br>
 +
. colony PCR for cons. pro. lock3crbs.GFP.DTT <br>
 +
. migrate cpromoter BBa_E0240 to pSB3K3 with mta key <br>
 +
. inoculation of B0034-GFP-DTT <br>
 +
. inoculation of Sherlock-GFP-DTT <br>
 +
. sequencing for sherlock in pSB1C3 <br>
 +
. digestion of B0034-GFP-DTT <br>
 +
. digestion of sherlock-GFP-DTT <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 3</b> <br>
 +
Wet lab<br>
 +
. digestion of P<sub>bad</sub> MTA dTT <br>
 +
. digestion of P<sub>bad</sub> dTT <br>
 +
. digestion of BBa_J23102 MCR GFP dTT <br>
 +
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
 +
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
 +
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
 +
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
 +
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> dTT <br>
 +
. Plasmid PCR for SherLock-GFP-DTT <br>
 +
. ligation of B0034-GFP-DTT <br>
 +
. ligation of SherLock-GFP-DTT <br>
 +
. sequencing <br>
 +
. check plate for arabinose induced mcr mta while singing <br>
 +
. extraction of key and lock <br>
 +
. restriction check for key with lock <br>
 +
. extraction of key1 DTT <br>
 +
. ligation of key1 DTT with P<sub>bad</sub> <br>
 +
. Gel check for O/N colonyPCR <br>
 +
. PCR check <br>
 +
. Check 031 <br>
 +
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br>
 +
. transform lock3c RBS.GFP.dTT <br>
 +
. Midi-prep preparations <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 4</b> <br>
 +
Wet lab<br>
 +
. Gel purification of digested insert and backbone <br>
 +
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
 +
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
 +
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
 +
. Transformation of ligated projects mentioned above <br>
 +
. Colony PCR for sherLock-GFP-DTT <br>
 +
. Colony PCR for B0034-GFP-DTT <br>
 +
. Inoculation of successful colonies <br>
 +
. Gel check for O/N Colony PCR <br>
 +
. Gel check for O/N PCR <br>
 +
. Gel check for Erics restriction check <br>
 +
. Inoculation of successful colonies <br>
 +
. Midi-prep preparations <br>
 +
. Gel purification <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 5</b> <br>
 +
Wet lab<br>
 +
. miniprep/ glycerol stock <br>
 +
. Transform P<sub>bad</sub> dTT <br>
 +
. Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 8</b> <br>
 +
Wet lab<br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·   <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 9</b> <br>
 +
Wet lab<br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·   <br>
 +
·      <br>
 +
·      <br>
 +
 
 +
<br><b>Sept 10</b> <br>
 +
Wet lab<br>
 +
. colony PCR <br>
 +
. ligations for final constructs <br>
 +
. inoculations <br>
 +
. P<sub>bad</sub>.lost your key.DTT <br>
 +
. sherlock <br>
 +
. gel check for colony PCR <br>
 +
. colony PCR <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 11</b> <br>
 +
Wet lab<br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·   <br>
 +
 
 +
<br><b>Sept 12</b> <br>
 +
Wet lab<br>
 +
. colony PCR <br>
 +
. restrict key1.DTT for P<sub>bad</sub> insertion <br>
 +
. ligate P<sub>bad</sub> with key1.DTT <br>
 +
. transform key1.dTT <br>
 +
. extract pSB3K3 <br>
 +
. Prepare TB <br>
 +
. Prepare dextrose for SOC <br>
 +
. check sequencing results <br>
 +
. Digestion of CLON041 for constructs <br>
 +
. ligation <br>
 +
. inoculation <br>
 +
. Digestion of CLON 036 <br>
 +
. colony PCR mcr mta <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 15</b> <br>
 +
Wet lab<br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·   <br>
 +
 
 +
 
 +
<br><b>Sept 16</b> <br>
 +
Wet lab<br>
 +
. mini prep P<sub>bad</sub>.key1.DTT <br>
 +
. restriction check P<sub>bad</sub>.key1.DTT <br>
 +
. gel check for O/N c.PCR <br>
 +
. extract P<sub>bad</sub>.key1 <br>
 +
. inoculate every CR.TA construct <br>
 +
. ligate transform key 1 with locks <br>
 +
. miniprep (B1, B2, and B3) <br>
 +
. plasmid PCR B1, B2 and B3 using sense oligo and VR <br>
 +
. Overnight digestion for ones which give positive results <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 17</b> <br>
 +
Wet lab<br>
 +
. purify P<sub>bad</sub>.key1 <br>
 +
. ligate P<sub>bad</sub>key1 DTT with locks <br>
 +
. transform locks.P<sub>bad</sub>.key1.DTT <br>
 +
. 2xYT (agar) AMP <br>
 +
. transform all <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 18</b> <br>
 +
Wet lab<br>
 +
. Spread a plate <br>
 +
. miniprep for 3 inoculations <br>
 +
. Sense oligo and VR PCR for HKUST Lock-DTT construct <br>
 +
. Overnight digestion of plasmid which gives a band around 1300bp <br>
 +
. EtoHpp for sequencing <br>
 +
. Miniprep constructs for submission <br>
 +
. inoculation <br>
 +
. transform 031, 034 <br>
 +
. Check "Diagonal constructs" and controls <br>
 +
. migrate CR constructs into pSB3K3 <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 19</b> <br>
 +
Wet lab<br>
 +
. miniprep lock.key candidate <br>
 +
. ligate problematic cr.key <br>
 +
. restriction check sherlock <br>
 +
. gel extraction<br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 20</b> <br>
 +
Wet lab<br>
 +
. miniprep CR.TA <br>
 +
. Colony PCR for CR.TA <br>
 +
. Colony PCR for CR.TA orthogonality <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 21</b> <br>
 +
Wet lab<br>
 +
. miniprep matching CR.TA <br>
 +
. Restriction check by EcoR1 and Pst1 <br>
 +
 
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 22</b> <br>
 +
Wet lab<br>
 +
. Run Gel check for matching set of CR.TA <br>
 +
. ligate problematic sets <br>
 +
. transform problematic sets <br>
 +
. prepare DNA for submission <br>
 +
. Gel purification <br>
 +
. Ligation lock-DTT with constitutive promoter <br>
 +
. transformation <br>
 +
. pSB3K3-CRs ligation <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 23</b> <br>
 +
Wet lab<br>
 +
. pSB3K3-CRs transformation <br>
 +
. Gel extractions <br>
 +
. Gel purifications <br>
 +
. TB <br>
 +
. Agar L (+) plates <br>
 +
. Send DNA to BGI for sequencing <br>
 +
. C. PCR <br>
 +
. Inoculate possible candidates <br>
 +
. ligate problematic sets <br>
 +
. transform confirmed constructs <br>
 +
. inoculation for HKUST lock Construct <br>
 +
. colony PCR For lock <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 24</b> <br>
 +
Wet lab<br>
 +
. Competent cells preparations <br>
 +
. minipreps <br>
 +
. colony PCR <br>
 +
. miniprep for Biobrick submission <br>
 +
. send plasmid for sequencing <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 25</b> <br>
 +
Wet lab<br>
 +
. insane prep <br>
 +
. restriction check <br>
 +
. Gel check for previous PvuII digestions <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 26</b> <br>
 +
Wet lab<br>
 +
. Gel check PvuII digestions <br>
 +
. Gel check AseI digestions <br>
 +
. 3A assembly for non pSB1C3 <br>
 +
. final batch of ligation for all missing component <br>
 +
. prepare DNA for submission <br>
 +
. Extraction for ligation <br>
 +
. Checking constructs-3 constructs using digestion check <br>
 +
. transformation for LOCK-8 <br>
 +
. Restriction check for controls for MCR/MTA <br>
 +
. Transformation of confirmed plasmid <br>
 +
 
 +
 
 +
<br><b>Sept 27</b> <br>
 +
Wet lab<br>
 +
. C. PCR for H series... <br>
 +
. inoculate possible candidates <br>
 +
. check self ligated pSB3K3 <br>
 +
. Gel check for possible candidates AseI <br>
 +
. Gel check for possible candidates PvuII <br>
 +
. Ligated H6 H7 <br>
 +
. transformed H6 H7 <br>
 +
. O/N inoculation of MCR/MTA for characterization <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 28</b> <br>
 +
Wet lab<br>
 +
. Extract pSB1C3 <br>
 +
. Extract 036 <br>
 +
. miniprep "H" series candidates <br>
 +
. Repeat CHL restrictions <br>
 +
. Repeat CHL ligations <br>
 +
. Repeat CHL transformations <br>
 +
. Gel check for "7" series using NcoI <br>
 +
. restriction check AseI <br>
 +
. restriction check NcoI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization. <br>
 +
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Sept 29</b> <br>
 +
Wet lab<br>
 +
. restriction check AseI <br>
 +
. restriction check NcoI <br>
 +
. mini prep 6 + MCR/MTA (thanks from Doyle) <br>
 +
. Restriction check for MCR/MTA in pSB3K3. <br>
 +
. restrict 036 <br>
 +
. purify 036 <br>
 +
. ligate monkey with sherlock <br>
 +
. Gel check for NcoI and AseI <br>
 +
. Get pSB1C3 backbone EcoRI and PstI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
 +
. inoculate Lock and Keys for crude functional assay. <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 30</b> <br>
 +
Wet lab<br>
 +
. miniprep 11 tubes in shaker c <br>
 +
. restriction check H" series with NcoI <br>
 +
. Restrict inoculated colonies wiht AseI <br>
 +
. Restrict inoculated colonies wiht NcoI <br>
 +
. Gel purifications <br>
 +
. ligation of H series and some from other series... <br>
 +
. transformation of 16 confirmed constructs <br>
 +
 
 +
 
 +
<br><b>Oct 1</b> <br>
 +
Wet lab<br>
 +
. digestion of key3,HKUSTkey<br>
 +
. miniprep- final constructs <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
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Latest revision as of 00:38, 18 October 2014



Notebook

Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately.






Pneumosensor

June 2014

Week 1
Week 2
Week 3
Week 4

July 2014

Week 1
Week 2
Week 3
Week 4
Week 5

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5





Riboregulator

July 2014

Week 1
Week 2
Week 3
Week 4

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5

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Home

Pneumosensor

Riboregulator

Human Practice

Team

WetLab

Achievement

Retrieved from "http://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/notebook"