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| − | {{Freiburg/Menubar}}
| + | /********************************* WIKI SETTINGS ********************************/ |
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| − | <h5 style="text-align: center"> Our Protocols </h5>
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| − | <a href="https://2016.igem.org/Team:Freiburg/NotebookCloning"> <img class="imga" src="https://static.igem.org/mediawiki/2016/1/14/T--Freiburg--CloningLab.png" alt="Smiley face"></a>
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| + | /********************************* CONTENT OF THE PAGE ********************************/ |
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| − | <!-- Click on each image to get directly to the method description of each group. -->
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| − | </center>
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| − | </div>
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| | + | /*LAYOUT */ |
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| − | <div class="color8">
| + | .full_size { |
| − | <div class="para_center_20">
| + | width: 100%; |
| − | <div class="level1"> | + | } |
| − | <h1 class="sectionedit1"><a name="lab_book_cloning" id="lab_book_cloning">Lab Book Cloning</a></h1>
| + | |
| − | <div class="tags"> To keep an overview of the cloned constructs every plasmid was assigned to an ID: pIG16_000.
| + | |
| − | <br/> All used oligos were assigned to an ID as well: oIG16_000.
| + | |
| − | <br/> The complete list of the resulting bacterial strains and oligos can be found in the attached Excel files. The spore coat proteins cotZ, cotG, cotB and cgeA were amplified from the genome of B. subtilis 168. The anti-GFP nanobody and the GST were amplified from plasmids provided by Dr. Nicole Gensch and Dr. Maximilian Ulbrich. For the cloning strategy see Project - <a target="_blank" href='https://2016.igem.org/Team:Freiburg/Goals_Approach' >Approach</a>.
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="color2">
| + | .full_size img { |
| − | <div class="para_20">
| + | padding: 10px 15px; |
| − | <!-- EDIT1 SECTION "Lab Book Cloning" [1-595] -->
| + | width: 96.5%; |
| − | <h3 class="sectionedit2"><a name="section070716" id="section070716">07.07.16</a></h3>
| + | } |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Transformation</strong>
| + | |
| − | <br/> Julia Bartels from iGEM 2012 sent us integration vectors for B.subtilis. Sebastian from AG Weber (BIOSS) shared plasmids containing mCherry and GFP. </p>
| + | |
| − | <p> <strong>Vectors:</strong>
| + | |
| − | <br/> No.1. pBS0K-Pspac-[RFP]
| + | |
| − | <br/> No.2. pBS1C3-[RFP]
| + | |
| − | <br/> No.3. pBS2E-[RFP]
| + | |
| − | <br/> No.4. pBS3Clux-[RFP]
| + | |
| − | <br/> No.5. pBS4S-[RFP]
| + | |
| − | <br/> No.6. pSBBs4S-Sporovector
| + | |
| − | <br/>
| + | |
| − | <br/> 7. mCherry
| + | |
| − | <br/> 8. GFP
| + | |
| − | <br/>
| + | |
| − | <br/> Transformation of the Vectors from Julia Bartels and Sebastian (from AG Weber) into chemically competent E.coli K12 DH5alpha was performed according to protocol. The E.coli were incubated for 1 hour at 37 °C and 250 rpm and spread on LB-Agar plates supplemented with ampicilin. Incubation for o/n at 37 °C.
| + | |
| − | <br/>
| + | |
| − | <br/> <strong>2) Preparation of competent E.coli</strong>
| + | |
| − | <br/> For the preparation for chemically competent E.coli one colony from an agar plate was picked in inoculated in 5 mL LB-medium. Incubation o/n at 37 °C and 250 rpm.
| + | |
| − | <br/>
| + | |
| − | <br/> </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT2 SECTION "07.07.16" [596-1494] -->
| + | |
| − | <h3 class="sectionedit3"><a name="section080716" id="section080716">08.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Production of competent E.coli</strong>
| + | |
| − | <br/> Preparation of chemically competent cells according to the protocol of the manufacturer (Zymoresearch, Z competent Mix&Go kit)
| + | |
| − | <br/> The resulting cell suspension was aliquoted (100 µL) in tubes and stored at -80 °C.
| + | |
| − | <br/>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Inoculation </strong>
| + | |
| − | <br/> From each transformation plate(No. 1-6) 5 colonies were picked and inoculated in 5 mL LB medium supplemented with Ampicilin.
| + | |
| − | <br/> Incubation o/n at 37°C and 250 rpm.
| + | |
| − | <br/> The B.subtilis W168 strain was inoculated in 5 mL of LB medium and incubated o/n at 37 °C and 250 rpm. In parallel, a sample of the W168 strain was spread on a LB agar plate and incubated o/n at 37 °C.
| + | |
| − | <br/> </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT3 SECTION "08.07.16" [1495-2174] -->
| + | |
| − | <h3 class="sectionedit4"><a name="section090716" id="section090716">09.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> The the plasmids of the inoculated colonies (No.1-6) were prepared using the QiaGen MiniPrep kit according the instructions of the manufacturer and eluted in 20 µL of ultra pure water. The concentration of the DNA was spectroscopically determined by a Nanodrop.
| + | |
| − | <br/>
| + | |
| − | <br/> <strong>2) Colony PCR</strong> Colony PCR for the amplication of the cotZ and cgeA gene was performed using the following oligos:
| + | |
| − | <br/> cotZ: oIG16_1+2 Annealing temperature: 57 °C
| + | |
| − | <br/> cgeA: oIG16_43+34 Annealing temperature: 65 °C
| + | |
| − | <br/>
| + | |
| − | <br/> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit5">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1 B.subtilis colony</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">-</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">1.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">1.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">4.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad20 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT5 TABLE [2728-2921] -->
| + | |
| − | <p>
| + | |
| − | <br/> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit6">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 1 min</td>
| + | |
| − | <td class="col3">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">58 or 65</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3"> 30</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT6 TABLE [2946-3156] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
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| − | </div>
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| − | </div>
| + | |
| − | <!--green -->
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| − | </div>
| + | |
| − | <!-- para20 -->
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| − | <div class="color3">
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| − | <div class="para_20">
| + | |
| − | <!-- EDIT4 SECTION "09.07.16" [2175-3161] -->
| + | |
| − | <h3 class="sectionedit7"><a name="section100716" id="section100716">10.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Gel electrophoresis</strong>
| + | |
| − | <br/> 20 µL of the colony PCR samples were supplemented with the appropriate amount of 10X Orange G loading buffer, loaded on a 1 % agarose TAE gel and subjected to electrophoresis for 40 min at 120 V. The DNA was stained with Midori Green. (No bands were observable at all. The amount of Midori Green was not sufficient)
| + | |
| − | <br/> </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT7 SECTION "10.07.16" [3162-3530] -->
| + | |
| − | <h3 class="sectionedit8"><a name="section110716" id="section110716">11.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1)Colony PCR</strong>
| + | |
| − | <br/> from 09.07.2016 was repeated at a total volume of 50 µL. After electrophoresis the sample were supplemented with the appropriate amount of 10X orange G loading buffer and loaded on an 1 % TAE agarose gel. The gel was subjected to electrophoresis for 40 min at 120 V. The DNA was stained with Midori Green.
| + | |
| − | <br/> Only the amplified cotZ was visible at UV Light.<br/>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/1/12/T--Freiburg--CloningJournal1.png" > </center><br/> <br/>
| + | .half_size { |
| − |
| + | width: 50%; |
| | + | } |
| | | | |
| − | The cotZ band was excised and the DNA was extracted using the QiaQuick Gel extraction kit according the the instructions of the manufacturer. The DNA was eluted in 20 µL of ultra pure H2O and the concentration was photometrically determined by a NanoDrop.
| + | .half_size img { |
| − | <br/> CotZ gelextraction concentration: 18 ng/µL </p>
| + | padding: 10px 15px; |
| − | </div>
| + | width: 93%; |
| − | <!-- EDIT8 SECTION "11.07.16" [3531-4296] -->
| + | } |
| − | <h3 class="sectionedit9"><a name="section120716" id="section120716">12.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Colony PCR</strong>
| + | |
| − | <br/> Colony-PCR for the amplification of cgeA and cotZ was repeated
| + | |
| − | <br/> Primer:
| + | |
| − | <br/> cgeA: oIG16_034 + 043
| + | |
| − | <br/> cotZ: oIG16_001 + 002
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit10">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1 B.subtilis colony W168</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">-</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad50 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT10 TABLE [4486-4683] -->
| + | |
| − | <p>
| + | |
| − | <br/> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit11">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 1 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">58 or 65</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">30X </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT11 TABLE [4708-4917] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> The samples were supplemented with the appropriate amount of 10 X Orange G loading dye and loaded on a 1 % TAE agarose gel. As molecular marker a 2-log marker was used.
| + | |
| − | <br/> No bands were observed at UV light. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!--para20-->
| + | |
| − | </div>
| + | |
| − | <!--color-->
| + | |
| − | <div class="color4">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT9 SECTION "12.07.16" [4297-5133] -->
| + | |
| − | <h3 class="sectionedit12"><a name="section130716" id="section130716">13.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Gradient colony PCR</strong>
| + | |
| − | <br/> To amplify cgeA 7 samples (20 µL each) were amplified by gradient colony PCR applying annealing temperatures from 58 - 68 °C.
| + | |
| − | <br/> An additional 50µl sample was made to amplify CotZ.
| + | |
| − | <br/> </p>
| + | |
| − | <p> The samples were supplemented with 10 X Orange G loading buffer and analyzed by gel electrophoresis. No bands were observable.
| + | |
| − | <br/>
| + | |
| | | | |
| − | <center><img align="middle" style="width:600px" src="https://static.igem.org/mediawiki/2016/6/6d/T--Freiburg--CloningJournal2.png" > </center><br/>
| + | .clear { |
| | + | clear: both; |
| | + | } |
| | | | |
| − | <br/> <center><img class="something"src="https://static.igem.org/mediawiki/2016/8/8c/T--Freiburg--CloningJournal3.png" > </center><br/>
| + | .highlight { |
| | + | width: 90%; |
| | + | margin: auto; |
| | + | padding: 15px 5px; |
| | + | background-color: #f2f2f2; |
| | + | } |
| | | | |
| − | <br/> </p>
| + | .my_center { |
| − | <div class="wrap_column plugin_wrap" style="width:40%;">
| + | display: flex; |
| − | <p>
| + | justify-content: center; |
| | + | } |
| | | | |
| | + | .something { |
| | + | width: 500px; |
| | + | } |
| | | | |
| | + | .color0 { |
| | + | /* white */ |
| | + | background-color: white; |
| | + | } |
| | | | |
| − | <div class="wrap_column plugin_wrap" style="width:40%;"></div>
| + | .color1 { |
| − | </div>
| + | /* dark purple*/ |
| − | <!-- EDIT12 SECTION "13.07.16" [5134-5679] -->
| + | background-color: #32014f; |
| − | <h3 class="sectionedit13"><a name="section140716" id="section140716">14.07.16</a></h3>
| + | } |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Repeat of gradient colony PCR</strong>
| + | |
| − | <br/> To amplify cgeA 7 samples (20 µL each) were amplified by gradient colony PCR applying annealing temperatures from 58 - 68 °C. The duration of initial denaturation was elongated to 5 min.
| + | |
| − | <br/> The samples were supplemented with 10 X Orange G loading buffer and analyzed by gel electrophoresis. No bands were observable.
| + | |
| | | | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/c/c3/T--Freiburg--CloningJournal4.png" > </center><br/>
| + | .color1 h1, |
| | + | .color1 h2, |
| | + | .color1 h3, |
| | + | .color1 h3 a, |
| | + | .color1 h4, |
| | + | .color1 h4 a, |
| | + | .color1 h5, |
| | + | .color1 h5 a, |
| | + | .color1 h6, |
| | + | .color1 h6 a, |
| | + | .color1 div, |
| | + | .color1 strong, |
| | + | .color1 p { |
| | + | /*color: #bbd4d4;*/ |
| | + | color: whitesmoke; |
| | + | } |
| | | | |
| − | </p>
| + | .color2 { |
| − | </div>
| + | /*pinkish*/ |
| − |
| + | background-color: #871150; |
| − | <h3 class="sectionedit14"><a name="section150716" id="section150716">15.07.16</a></h3>
| + | /* color: #aaaaaa;*/ |
| − | <div class="level3">
| + | } |
| − | <p> <strong>1) Lysis of B. subtilis for colony PCR</strong>
| + | |
| − | <br/> 4 different approaches were tried to lyse the bacteria prior to colony PCR.
| + | |
| − | <br/> 1. <a href="https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf" class="urlextern" target="_Blank" title="https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf" rel="nofollow">https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf</a>
| + | |
| − | <br/> 2. <a href="http://www.scs.illinois.edu/rao/protocol-subtilis-colony.php" class="urlextern" target="_Blank" title="http://www.scs.illinois.edu/rao/protocol-subtilis-colony.php" rel="nofollow">http://www.scs.illinois.edu/rao/protocol-subtilis-colony.php</a>
| + | |
| − | <br/> 3. Mini-Prep lysis buffer.
| + | |
| − | <br/> 4. Laemmlie sample buffer 100°C 5 Min.
| + | |
| − | <br/> </p>
| + | |
| − | <p> 1 µl of each sample was used for amplification of cotZ using the oligos oIG16_1+2. The extracted cotZ gene from 11.07.16 was amplified as control alongside the lysed samples
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit15">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">lysed B.subtilis W168</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad50 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT15 TABLE [6709-6907] -->
| + | |
| − | <p>
| + | |
| − | <br/> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit16">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">65</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3"> 30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3"> 30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT16 TABLE [6932-7135] -->
| + | |
| | | | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/5/55/T--Freiburg--CloningJournal5.png" > </center><br/>
| + | .color2 h1, |
| − |
| + | .color2 h2, |
| − | <p> <strong> 2) Testdigestion</strong>
| + | .color2 h3, |
| | + | .color2 h3 a, |
| | + | .color2 h4, |
| | + | .color2 h4 a, |
| | + | .color2 h5, |
| | + | .color2 h5 a, |
| | + | .color2 h6, |
| | + | .color2 h6 a, |
| | + | .color2 div, |
| | + | .color2 strong, |
| | + | .color2 p { |
| | + | /*color: #aaaaaa;*/ |
| | + | color: white; |
| | + | } |
| | | | |
| − | <br/> PvuII test digestion for verification of the plasmids sent by Julia Bartels.
| + | .color3 { |
| − | <br/> Reaction conditions:
| + | /*orange*/ |
| − | <br/> </p>
| + | background-color: #FFB60E; |
| − | <div class="table sectionedit17">
| + | /* color: #d25852;*/ |
| − | <table class="inline">
| + | } |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">MiniPrep DNA</td>
| + | |
| − | <td class="col1">200-600 ng/µL</td>
| + | |
| − | <td class="col2">2 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">CutSmart Buffer (NEB)</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PvuII-HF</td>
| + | |
| − | <td class="col1">20 U/ µL</td>
| + | |
| − | <td class="col2">0.1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT17 TABLE [7320-7470] -->
| + | |
| − | <p> Incubation at 37 °C for 1 hour. The samples were analyzed by gel electrophoresis on a 1 % TAE agarose gel.
| + | |
| − | <br/> </p>
| + | |
| − |
| + | |
| − | <p>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/6/61/T--Freiburg--CloningJournal6.png" > </center><br/>
| + | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/7/7c/T--Freiburg--CloningJournal7.png" > </center><br/>
| + | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/3/34/T--Freiburg--CloningJournal8.png" > </center><br/>
| + | |
| − | <br/>
| + | |
| − | <br/>
| + | |
| − | <br/> <strong>3) Subcloning</strong>
| + | |
| − | <br/> Subcloning of the amplified <em>cotZ</em> gene into the linearized pJET1.2 vector was performed using the pJET subcloning kit according to the instructions of the manufacturer. 5 µL of the ligation mixture was used for transformation of chemically competent E.coli DH5alpha (Mix & Go). The transformed bacteria were spread on a LB-agar plate supplemented with amplicilin and incubated o/n at 37 °C. A control ligation was prepared using the DNA fragments supplied by the manufacturer.
| + | |
| − | <br/> </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!--para20-->
| + | |
| − | </div> | + | |
| − | <!--color-->
| + | |
| − | <div class="color5">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT14 SECTION "15.07.16" [6129-8336] -->
| + | |
| − | <h3 class="sectionedit18"><a name="section160716" id="section160716">16.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Colony PCR </strong>
| + | |
| − | <br/> for the amplication of the cgeA was performed using the following oligos:
| + | |
| − | <br/> cgeA: oIG16_43+34 Annealing temperature: 65 °C
| + | |
| − | <br/> To lyse the bacteria prior to the colony PCR.
| + | |
| − | <br/> <a href="https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf" class="urlextern" target="_Blank" title="https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf" rel="nofollow">https://static.igem.org/mediawiki/2015/b/bf/Technion_Israel_2015_Colony_PCR_for_B.Subtilis_.pdf</a>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit19">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">lysed B.subtilis W168</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad50 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT19 TABLE [8670-8869] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit20">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">65</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT20 TABLE [8895-9095] -->
| + | |
| − | <p>
| + | |
| − | <br/>
| + | |
| − | <br/> <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/01/T--Freiburg--CloningJournal9.png" > </center><br/>
| + | |
| | | | |
| − | <br/>
| + | .color3 h1, |
| − | <br/>
| + | .color3 h2, |
| − | <br/>
| + | .color3 h3, |
| − | <br/> <strong>2) Inoculation</strong>
| + | .color3 h3 a, |
| − | <br/> Subcloning and transformation of pJET1.2-cotZ resulted only in a few colonies. Three of them were picked and inoculated in 5 mL LB-medium w/ ampicilin and incubated o/n at 37°C and 250 rpm. The transformation with the control plate yielded >100 colonies. </p>
| + | .color3 h4, |
| − | </div>
| + | .color3 h4 a, |
| − | <!-- EDIT18 SECTION "16.07.16" [8337-9457] -->
| + | .color3 h5, |
| − | <h3 class="sectionedit21"><a name="section170716" id="section170716">17.07.16</a></h3>
| + | .color3 h5 a, |
| − | <div class="level3">
| + | .color3 h6, |
| − | <p> <strong>1) Colony PCR</strong>
| + | .color3 h6 a, |
| − | <br/> 5 µL of vegetative B.subtilis from a liquid culture were diluted 1:10 in Qiagen EB buffer and incubated at 100°C for 5min to achieve cell lysis
| + | .color3 div, |
| − | <br/> 1 µL of the dilution was used for amplification of cgeA by gradient PCR and 8 samples were prepared.
| + | .color3 strong, |
| − | <br/> </p>
| + | .color3 p { |
| − | <p> <strong> Reaction conditions</strong>
| + | /*color: #d25852;*/ |
| − | <br/> </p>
| + | color: black; |
| − | <div class="table sectionedit22">
| + | } |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">lysed B.subtilis W168</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad20 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT22 TABLE [9778-9971] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit23">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">gradient 58-68</td>
| + | |
| − | <td class="col2">20 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">10</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT23 TABLE [9997-10210] -->
| + | |
| − | <p> After thermal cycling the samples were supplemented with the appropriate amount of 10X Orange G loading dye and analyed by agarose gel electrophoresis. </p>
| + | |
| − | <p> <br/>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/9/9f/T--Freiburg--CloningJournal10.png" > </center><br/>
| + | .color4 { |
| | + | /*greenish*/ |
| | + | background-color: #168152; |
| | + | /* color: #5d995d;*/ |
| | + | } |
| | | | |
| − | </p>
| + | .color4 h1, |
| − | <p> <strong>2) MiniPrep</strong>
| + | .color4 h2, |
| − | <br/> The plasmids pJET1.2-cotZ were prepared using the QIAprep Spin Miniprep Kit according the instructions of the manufacturer and eluted in 50µL of EB Buffer. The concentration of the DNA was spectroscopically determined by a nanodrop.
| + | .color4 h3, |
| − | <br/> </p>
| + | .color4 h3 a, |
| − | <div class="table sectionedit24">
| + | .color4 h4, |
| − | <table class="inline">
| + | .color4 h4 a, |
| − | <tr class="row0">
| + | .color4 h5, |
| − | <th class="col0">DNA</th>
| + | .color4 h5 a, |
| − | <th class="col1">concentration[ng/µL]</th>
| + | .color4 h6, |
| − | </tr>
| + | .color4 h6 a, |
| − | <tr class="row1">
| + | .color4 div, |
| − | <td class="col0">pJet_cotZ_#1</td>
| + | .color4 strong, |
| − | <td class="col1">120</td>
| + | .color4 p { |
| − | </tr>
| + | /*color: #5d995d;*/ |
| − | <tr class="row2">
| + | color: white; |
| − | <td class="col0">pJet_cotZ_#2</td>
| + | } |
| − | <td class="col1">89</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pJet_cotZ_#3</td>
| + | |
| − | <td class="col1">68</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT24 TABLE [10699-10781] -->
| + | |
| − | <p>
| + | |
| − | <br/> <strong>3) Testdigestion</strong>
| + | |
| − | <br/> <strong> Reaction conditions </strong> </p>
| + | |
| − | <div class="table sectionedit25">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">68 - 120 ng/µL</td>
| + | |
| − | <td class="col2">7 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">11 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT25 TABLE [10834-10969] -->
| + | |
| − | <p> Incubation for 1 h at 37 °C. Analysis of digestion by agarose gel electrophoresis. </p>
| + | |
| − | <p> <br/>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/b8/T--Freiburg--CloningJournal11.png" > </center><br/>
| + | .color5 { |
| | + | /*bluish*/ |
| | + | background-color: #3d4d60; |
| | + | /* color: #95a5a6;*/ |
| | + | } |
| | | | |
| − | 2-log DNA ladder is bad in the last couple of gels –> discarded.
| + | .color5 h1, |
| − | <br/> Not the expected band pattern. Subcloning should be repeated.
| + | .color5 h2, |
| − | <br/> </p>
| + | .color5 h3, |
| − | <p> <strong>3) Colony PCR</strong>
| + | .color5 h3 a, |
| − | <br/> 5 µL of vegetative B.subtilis from a liquid culture were diluted 1:10 in Qiagen EB buffer and incubated at 100°C for 5min to achieve cell lysis
| + | .color5 h4, |
| − | <br/> 1 µL of the dilution was used for amplification of cgeA and cotZ by PCR.
| + | .color5 h4 a, |
| − | <br/> oIG16_001+002: CotZ, Tm = 58 °C oIG16_034+043: cgeA, Tm = 63 °C </p>
| + | .color5 h5, |
| − | <div class="table sectionedit26">
| + | .color5 h5 a, |
| − | <table class="inline">
| + | .color5 h6, |
| − | <tr class="row0">
| + | .color5 h6 a, |
| − | <th class="col0">component</th>
| + | .color5 div, |
| − | <th class="col1">concentration</th>
| + | .color5 strong, |
| − | <th class="col2">volume [µL]</th>
| + | .color5 p { |
| − | </tr>
| + | /*color: #95a5a6;*/ |
| − | <tr class="row1">
| + | color: white; |
| − | <td class="col0">lysed B.subtilis W168</td>
| + | } |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">dNTPs</td>
| + | |
| − | <td class="col1">40 µM</td>
| + | |
| − | <td class="col2">0.25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">HF Buffer</td>
| + | |
| − | <td class="col1">5X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Phusion Pol</td>
| + | |
| − | <td class="col1">2U/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ddH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad50 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT26 TABLE [11578-11777] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Cycler Program</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit27">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">58 or 63</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">30X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT27 TABLE [11803-12009] -->
| + | |
| − | <p>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/07/T--Freiburg--CloningJournal12.png" ></center>
| + | .color6 { |
| − | </p>
| + | /*blue*/ |
| − | <p> The bands were excised from the gel and stored at -20 °C o/n </p>
| + | background-color: #09305f; |
| − | </div>
| + | /* color: #aea8d3;*/ |
| − | <!-- EDIT21 SECTION "17.07.16" [9458-12146] -->
| + | } |
| − | <h3 class="sectionedit28"><a name="section180716" id="section180716">18.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Gelextraction & Subcloning</strong>
| + | |
| − | <br/> The DNA was extracted using the QiaGen gel extraction kit according to the protocol of the manufacturer and eluted in 30 µL of ultra pure water. The concentration was determined spectroscopically by a nanodrop.
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit29">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">gene</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">cgeA</td>
| + | |
| − | <td class="col1">6 ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">cotZ#1</td>
| + | |
| − | <td class="col1">8 ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">cotZ#2</td>
| + | |
| − | <td class="col1">11 ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT29 TABLE [12419-12492] -->
| + | |
| − | <p> The extracted DNA was subcloned into a linearized pJET1.2 vector using the CloneJET PCR Cloning Kit according to the protocol of the manufacturer. 5 µL of the ligation mixture were transformed into chemically competent E.coli DH5alpha (Mix&Go) and spread on a LB-agar plate supplemented with ampicilin and incubated at 37 °C o/n. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!--para20-->
| + | |
| − | </div> | + | |
| − | <!--color-->
| + | |
| − | <div class="color6">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT28 SECTION "18.07.16" [12147-12827] -->
| + | |
| − | <h3 class="sectionedit30"><a name="section190716" id="section190716">19.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculation</strong>
| + | |
| − | <br/> 4 colonies per plate were picked and incubated in 5 mL LB-medium (amp) o/n at 37 °C and 250 rpm. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT30 SECTION "19.07.16" [12828-12966] -->
| + | |
| − | <h3 class="sectionedit31"><a name="section200716" id="section200716">20.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> The plasmids pJET1.2-cotZ-I.#1-4;pJET1.2-cotZ-II.#1-4 and pJET1.2-cgeA#1-4 were prepared using the QIAprep Spin Miniprep Kit according the instructions of the manufacturer and eluted in 20µL of ultra pure water (for better results put tubes for 2 minutes on 50°C thermo; centrifuge for 1min at 13000rpm and repeat with 10µL; increases the amount of deluted DNA). The concentration of the DNA was spectroscopically determined by a nanodrop.
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit32">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">DNA</th>
| + | |
| − | <th class="col1">concentration[ng/µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pJet1.2_cgeA_#1</td>
| + | |
| − | <td class="col1">127</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pJet1.2_cgeA_#2</td>
| + | |
| − | <td class="col1">214 –> Seq</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pJet1.2_cgeA_#3</td>
| + | |
| − | <td class="col1">279</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">pJet1.2_cgeA_#4</td>
| + | |
| − | <td class="col1">141</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">pJet1.2_cotZ-I_#1</td>
| + | |
| − | <td class="col1">260</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">pJet1.2_cotZ-I_#2</td>
| + | |
| − | <td class="col1">101</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">pJet1.2_cotZ-I_#3</td>
| + | |
| − | <td class="col1">409</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">pJet1.2_cotZ-I_#4</td>
| + | |
| − | <td class="col1">435</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">pJet1.2_cotZ-II_#1</td>
| + | |
| − | <td class="col1">475</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">pJet1.2_cotZ-II_#2</td>
| + | |
| − | <td class="col1">350 –> Seq</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0">pJet1.2_cotZ-II_#3</td>
| + | |
| − | <td class="col1">353</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row12">
| + | |
| − | <td class="col0">pJet1.2_cotZ-II_#4</td>
| + | |
| − | <td class="col1">342</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT32 TABLE [13449-13776] -->
| + | |
| − | <p>
| + | |
| − | <br/> <strong>2) Test digestion</strong>
| + | |
| − | <br/> Due to the concentraion difference of the DNA the samples where divided into two groups </p>
| + | |
| − | <p> <strong> Reaction conditions </strong>
| + | |
| − | <br/> DNA concentration < 300ng/µL
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit33">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">101 - 279 ng/µL</td>
| + | |
| − | <td class="col2">5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">14.1 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT33 TABLE [13954-14092] -->
| + | |
| − | <p>
| + | |
| − | <br/> DNA concentration > 300ng/µL
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit34">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">342-475 ng/µL</td>
| + | |
| − | <td class="col2">1.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20U/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">12.6 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT34 TABLE [14128-14266] -->
| + | |
| − | <p>
| + | |
| − | <br/> Control group < 300ng/µL
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit35">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">101 - 279 ng/µL</td>
| + | |
| − | <td class="col2">5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">5 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT35 TABLE [14298-14374] -->
| + | |
| − | <p>
| + | |
| − | <br/> Control group > 300ng/µL
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit36">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">342-475 ng/µL</td>
| + | |
| − | <td class="col2">1.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">8,5 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT36 TABLE [14406-14484] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> Incubation for 1 h at 37 °C. Analysis of digestion by electrophoresis on a 1% TAE agarose gel.
| + | |
| − | <br/> </p>
| + | |
| − | <p>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/3/30/T--Freiburg--CloningJournal13.png" ></center> </p>
| + | .color6 h1, |
| | + | .color6 h2, |
| | + | .color6 h3, |
| | + | .color6 h3 a, |
| | + | .color6 h4, |
| | + | .color6 h4 a, |
| | + | .color6 h5, |
| | + | .color6 h5 a, |
| | + | .color6 h6, |
| | + | .color6 h6 a, |
| | + | .color6 div, |
| | + | .color6 strong, |
| | + | .color6 p { |
| | + | /*color: #aea8d3;*/ |
| | + | color: white; |
| | + | } |
| | | | |
| − | <p> <strong>3) Sequencing</strong>
| + | .color7 { |
| − | <br/> Sequencing of pJET1.2-cgeA #2 (IC0225) and pJET1.2-cotZII #2 (IC0224) by GATC biotech. </p>
| + | /*dark red*/ |
| − | </div>
| + | background-color: #5F0000; |
| − | <!-- EDIT31 SECTION "20.07.16" [12967-14775] -->
| + | /* color: #d24d57;*/ |
| − | <h3 class="sectionedit37"><a name="section210716" id="section210716">21.07.16</a></h3>
| + | } |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculation</strong>
| + | |
| − | <br/> Confirmation of the proper sequences for pJET1.2_cgeA#2 and pJET1.2_cotZII#2. The colonies were re-inoculated in 5 mL LB-medium (w/ amp) and incubated at 37 °C, 250 rpm o/n. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!--para20-->
| + | |
| − | </div> | + | |
| − | <!--color-->
| + | |
| − | <div class="color7">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT37 SECTION "21.07.16" [14776-14991] -->
| + | |
| − | <h3 class="sectionedit38"><a name="section220716" id="section220716">22.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Transformation & Inoculation</strong>
| + | |
| − | <br/> Nicole provided us with a LB-plate containing E.coli harboring pGEX6P1 and pRP261 (Both plasmids contain glutathion S transferase). Max provided us with a sample of pET303_aGFPnano_TEV_10His (plasmid containing the anti-GFP nanobody). The pET303 plasmid was transformed into chemically competent E.coli DH5alpha and spread on a LB-agar plate supplemented with ampicilin and incubated o/n at 37 °C. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT38 SECTION "22.07.16" [14992-15450] -->
| + | |
| − | <h3 class="sectionedit39"><a name="section230716" id="section230716">23.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculation</strong>
| + | |
| − | <br/> Inoculation of the E.coli DH5alpha containing pGEX6P1, pRP261 and pET303_aGFPnano in 5 mL LB medium supplemented with ampicilin. Incubation o/n at 37 °C, 250 rpm.
| + | |
| − | <br/> Glycerol stocks of pJET1.2-cgeA and pJET1.2-cotZ were prepared (15% [v/v] Glycerol).
| + | |
| − | <br/> Inoculation of pBS1C3-[RFP] culture #2 for test-transformation of B.subtilis. </p>
| + | |
| − | <p> <strong>2) Transformation</strong>
| + | |
| − | <br/> The pSB1C3-[RFP] vector from the iGEM distribution kit (plate 1, position 23-O) was solubilized with 10 µL of ultra pure water. 1 µL was used for transformation of chemically competent E.coli DH5alpha and spread on a LB-agar plate supplemented with chloramphenicol and incubated o/n at 37 °C. </p>
| + | |
| − | <p> <strong>3) Extension PCR </strong>
| + | |
| − | <br/> of cgeA (from pJET1.2_cgeA) and anti GFP-Nanobody (pET303) *Reaction conditions*
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit40">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">~10 ng/µL</td>
| + | |
| − | <td class="col2">1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Q5 High-Fidelity Master Mix (NEB)</td>
| + | |
| − | <td class="col1">2x</td>
| + | |
| − | <td class="col2">25 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure H2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT40 TABLE [16252-16428] -->
| + | |
| − | <div class="wrap_column plugin_wrap" style="width:47%;">
| + | |
| − | <p> Touchdown-PCR Template: pJET1.2_cgeA
| + | |
| − | <br/> Primer:olG16_44fw + olG16_45rw </p>
| + | |
| − | <p> <strong>Cycler Program GFP-Nanobody</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit41">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">72* (-1°C per cycle)</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">63</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT41 TABLE [16556-16853] -->
| + | |
| − | <p> Template: pET303_aGFPnano
| + | |
| − | <br/> Primer:olG30_fw/olG31_rw
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Cycler Program CgeA</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit42">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">72* (-1°C per cycle) </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72</td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">62</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT42 TABLE [16937-17234] -->
| + | |
| − | </div>
| + | |
| − | <div class="wrap_column plugin_wrap" style="width:47%;">
| + | |
| − | <p> After amplification the samples were analyzed by agarose gel electrophoresis. </p>
| + | |
| − | <p> <strong>Gel GFP-Nanobody/CgeA</strong> </p>
| + | |
| − | <p>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/4/41/T--Freiburg--CloningJournal14.png" ></center>
| + | .color7 h1, |
| | + | .color7 h2, |
| | + | .color7 h3, |
| | + | .color7 h3 a, |
| | + | .color7 h4, |
| | + | .color7 h4 a, |
| | + | .color7 h5, |
| | + | .color7 h5 a, |
| | + | .color7 h6, |
| | + | .color7 h6 a, |
| | + | .color7 div, |
| | + | .color7 strong, |
| | + | .color7 p { |
| | + | /*color: #d24d57;*/ |
| | + | color: white; |
| | + | } |
| | | | |
| − | </p>
| + | .color8 { |
| − | </div>
| + | /* do not change this color */ |
| − | <p>
| + | /*blue*/ |
| − | <br/> </p>
| + | background-color: #008783; |
| − | </div>
| + | /* color: #002a2a;*/ |
| − | </div>
| + | } |
| − | <!--para20-->
| + | |
| − | </div> | + | |
| − | <!--color-->
| + | |
| − | <!-- EDIT39 SECTION "23.07.16" [15451-17446] --> | + | |
| − | <div class="color8">
| + | |
| − | <div class="para_20">
| + | |
| − | <h3 class="sectionedit43"><a name="section240716" id="section240716">24.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Gelextraction</strong>
| + | |
| − | <br/> Gel Extraction of pET303-aGFPnanobody oIG16_30_fw/oIG16_031_rw PCR product.
| + | |
| − | <br/> 37.7ng/µl (18µl) stored at -20 °C
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) MiniPrep</strong>
| + | |
| − | <br/> Plasmid preparation of inoculated E.coli DH5alpha transformed with Mini prep of pBS1C3-RFP culture#2 using the QiaQuick MiniPrep kit according to the instructions of the manufacturer. Elution with 30 µL of ultra pure water.
| + | |
| − | <br/> 148.6ng/µl (28µl)
| + | |
| − | <br/> placed in freezer -20 </p>
| + | |
| − | <p>
| + | |
| − | <br/> Plasmid preparation of inoculated E.coli DH5alpha transformed with pGEX6P1, pRP261, pET303-aGFPnano_TEV_10His using the QiaQuick miniprep kit according to the instructions of the manufacturer. The plasmids were eluted in 30 µL of ultra pure water. The DNA concentration was determined by NanoDrop:
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit44">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Sample</th>
| + | |
| − | <th class="col1">concentration[ng/µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pRP261 #1</td>
| + | |
| − | <td class="col1">248.9</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pRP261 #2</td>
| + | |
| − | <td class="col1">175.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pRP261 #3</td>
| + | |
| − | <td class="col1">235.9</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">pRP261 #4</td>
| + | |
| − | <td class="col1">221.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">pGEX-6P1 #1</td>
| + | |
| − | <td class="col1">198.7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">pGEX-6P1 #2</td>
| + | |
| − | <td class="col1">177.3</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">pGEX-6P1 #3</td>
| + | |
| − | <td class="col1">149.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">pGEX-6P1 #4</td>
| + | |
| − | <td class="col1">131.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">pET303-aGFPnano_TEV_10His #1</td>
| + | |
| − | <td class="col1">167.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">pET303-aGFPnano_TEV_10His #2</td>
| + | |
| − | <td class="col1">151.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0">pET303-aGFPnano_TEV_10His #3</td>
| + | |
| − | <td class="col1">161.7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row12">
| + | |
| − | <td class="col0">pET303-aGFPnano_TEV_10His #4</td>
| + | |
| − | <td class="col1">165.0</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT44 TABLE [18202-18532] -->
| + | |
| − | <p> <strong>2) Testdigestion</strong>
| + | |
| − | <br/> Verification of the plasmid was performed by test digestion with EcoRI and PstI.
| + | |
| − | <br/> Reaction conditions:
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit45">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">131-248ng/µL</td>
| + | |
| − | <td class="col2">3 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT45 TABLE [18663-18815] -->
| + | |
| − | <p> The samples were incubated at 37 °C for 1h and analyzed by gel electrophoresis. </p>
| + | |
| − | <p>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/ff/T--Freiburg--CloningJournal15.png" ></center>
| + | .color8 h1, |
| | + | .color8 h2, |
| | + | .color8 h3, |
| | + | .color8 h3 a, |
| | + | .color8 h4, |
| | + | .color8 h4 a, |
| | + | .color8 h5, |
| | + | .color8 h5 a, |
| | + | .color8 h6, |
| | + | .color8 h6 a, |
| | + | .color8 div, |
| | + | .color8 strong, |
| | + | .color8 p { |
| | + | /*color: #002a2a;*/ |
| | + | color: white; |
| | + | } |
| | | | |
| − | </p>
| + | .color9 { |
| − | </div>
| + | /*lighter blue*/ |
| − | <!-- EDIT43 SECTION "24.07.16" [17447-18996] -->
| + | background-color: #146AD3; |
| − | <h3 class="sectionedit46"><a name="section250716" id="section250716">25.07.16</a></h3>
| + | color: #3e3e3e; |
| − | <div class="level3">
| + | } |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> Plasmids of pSB1C3 were prepared. DNA concentration was determined by Nanodrop:
| + | |
| − | <br/> Culture #1 127.5ng/µl
| + | |
| − | <br/> Culture #2 88.4ng/µl
| + | |
| − | <br/> Culture #3 116.8ng/µl
| + | |
| − | <br/> Culture #4 126.1ng/µl
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Testdigestion</strong> pSB1C3 was treated with </p>
| + | |
| − | <p> <strong>Digestion mixture:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit47">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">5-6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI-HF</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT47 TABLE [19290-19442] -->
| + | |
| − | <p> <strong>Agarose Gel of pSB1C3 digestion:</strong>
| + | |
| − | <br/>
| + | |
| − |
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/2/29/T--Freiburg--CloningJournal16.png" ></center>
| + | .color10 { |
| | + | /*lighter green/blue */ |
| | + | background-color: #00A4BE; |
| | + | color: #3e3e3e; |
| | + | } |
| | | | |
| | + | .color11 { |
| | + | /* blue*/ |
| | + | background-color: #1E4A7F; |
| | + | color: #3e3e3e; |
| | + | } |
| | | | |
| − | </p>
| + | .color12 { |
| − | <p> <strong>3) Inoculation</strong>
| + | /*pink*/ |
| − | <br/> Culture #3 was re-inoculated in chloramphenicol-medium. </p>
| + | background-color: #8C0D75; |
| − | </div>
| + | color: #3e3e3e; |
| − | <!-- EDIT46 SECTION "25.07.16" [18997-19629] -->
| + | } |
| − | <h3 class="sectionedit48"><a name="section260716" id="section260716">26.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> With culture #3 from the day before a standard mini prep was performed.
| + | |
| − | <br/> cultur #3 169,4ng/µl (28µl)
| + | |
| − | <br/> → placed in the -20 freezer </p>
| + | |
| − | <p> <strong>2) Colony PCR</strong>
| + | |
| − | <br/> Colony PCR of CotG and CotB from B. subtilis 168 genome. PCR for the amplication of the cotG and cgeB gene was performed using the following oligos:
| + | |
| − | <br/> cotG: oIG16_009+010 Annealing temperature: 62 °C
| + | |
| − | <br/> cotB: oIG16_017+018 Annealing temperature: 57 °C
| + | |
| − | <br/> Template DNA preparation: 1 µL of a o/n culture with B.subtilis W168 cells were diluted with EB-Buffer (Qiagen, 1:10) and incubated at 100°C for 5 min for lysis. 1 µL of the lysed cells was used as template for amplificaiton of the genes.
| + | |
| − | <br/>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Reaction conditions</strong> </p>
| + | |
| − | <div class="table sectionedit49">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Q5 HiFi MasterMix</td>
| + | |
| − | <td class="col1">2x</td>
| + | |
| − | <td class="col2">25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">lysed cells</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT49 TABLE [20355-20514] -->
| + | |
| − | <p> Appropriate controls w/o template DNA were included. </p>
| + | |
| − | <p> The PCR was analyzed by agarose gel electrophoresis.
| + | |
| − | <br/>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/3/3e/T--Freiburg--CloningJournal17.png" ></center>
| + | .img_menu { |
| | + | /*purple*/ |
| | + | background-color: rgb(67, 33, 79); |
| | + | padding: 20px; |
| | + | } |
| | | | |
| − | The bands corresponding to the size of the cotG and cotB were excised and the DNA was extracted using the QiaQuick Gel extraction kit according to the instructions of the manufacturer. The DNA was eluted in 30 µL of ultra pure water.
| + | .para_center_20 { |
| − | <br/> </p>
| + | font-size: 20px; |
| − | <p> <strong>2) Gel extraction</strong>
| + | text-align: justify; |
| − | <br/> The concentration of the extracted DNA was determined by a Nanodrop. </p>
| + | font-family: 'Open Sans', sans-serif; |
| − | <div class="table sectionedit50">
| + | padding: 2% 20% 2% 20%; |
| − | <table class="inline">
| + | letter-spacing: 2px; |
| − | <tr class="row0">
| + | line-height: 120%; |
| − | <th class="col0">sample</th>
| + | } |
| − | <th class="col1">concentration</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">CotB</td>
| + | |
| − | <td class="col1">50.2 ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">CotG</td>
| + | |
| − | <td class="col1">42.1 ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT50 TABLE [21055-21115] -->
| + | |
| − | <p> <strong>3) Subcloning</strong>
| + | |
| − | <br/> The amplified genes were subcloned into the pJET1.2 vector according to the protocol of the manufacturer, transformed into chemically competent E.coli DH5alpha, spread on a LB-agar plate supplemented with ampicilin and incubated o/n at 37 °C.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>4) Inoculation</strong>
| + | |
| − | <br/> The E.coli liquid cultures containing the plasmids pRP261, pGEX6P1 and pET303_aGFPnano_TEV_10His were reinoculated in 5 mL LB medium supplemented with ampicilin and incubated o/n at 37 °C and 250 rpm. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!-- para20-->
| + | |
| − | </div> | + | |
| − | <!-- color-->
| + | |
| − | <div class="color1"> | + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT48 SECTION "26.07.16" [19630-21607] -->
| + | |
| − | <h3 class="sectionedit51"><a name="section270716" id="section270716">27.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculation</strong>
| + | |
| − | <br/> 4 colonies of plated E.coli DH5alpha with the pJET1.2-cotB and pJET1.2-cotG plasmids were picked and inoculated in 5 mL of LB medium supplemented with ampicilin and incbated o/n at 37 °C and 250 rpm. <strong>2) Sequencing</strong>
| + | |
| − | <br/> Sequencing of plasmids sent by Julia Bartels: </p>
| + | |
| − | <div class="table sectionedit52">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Plasmid</th>
| + | |
| − | <th class="col1">colony#</th>
| + | |
| − | <th class="col2">Primer:oIG16_</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C-[RFP]</td>
| + | |
| − | <td class="col1">#2</td>
| + | |
| − | <td class="col2">025</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">026</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">028</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">029</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">032</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">pBS2E-[RFP]</td>
| + | |
| − | <td class="col1">#5</td>
| + | |
| − | <td class="col2">025</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">pBS4S-[RFP]</td>
| + | |
| − | <td class="col1">#5</td>
| + | |
| − | <td class="col2">025</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">26</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">27</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">SporoVector-[RFP]</td>
| + | |
| − | <td class="col1">#1</td>
| + | |
| − | <td class="col2">025</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">026</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row12">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">027</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT52 TABLE [21916-22115] -->
| + | |
| − | </div>
| + | |
| − | <!-- EDIT51 SECTION "27.07.16" [21608-22116] -->
| + | |
| − | <h3 class="sectionedit53"><a name="section280716" id="section280716">28.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Mini-prep:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> A standard Qiagen Mini-Prep was performed with the following cultures:
| + | |
| − | <br/> pJET1.2-cotB cultures #2/3/4, pJET1.2-cotG cultures #1/2/3/4, pGEX6P1 culture #1 (GST 2), pRP261 culture #1 (GST 1), pET303_aGFPnano_TEV_10His culture #1. </p>
| + | |
| − | <p> The DNA concentrations were measured with Nanodrop:
| + | |
| − | <br/> pJET1.2-cotB cultures #2=450,9(ng/µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #3=820,3(ng/µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #4=621,2(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #1=696,5(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #2=149,6(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #3=614,8(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #4=145,2(ng/µl)
| + | |
| − | <br/> pGEX6P1 culture #1=243,1(ng/µl)
| + | |
| − | <br/> pRP261 culture #1=308,2(ng/µl)
| + | |
| − | <br/> pET303_aGFPnano_TEV_10His culture #1=91,1(ng/µl)
| + | |
| − | <br/> </p>
| + | |
| − | <p> µl taken for Test Digestion:
| + | |
| − | <br/> pJET1.2-cotB cultures #2=1(µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #3=1(µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #4=1(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #1=1(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #2=4(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #3=1(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #4=4(µl)
| + | |
| − | <br/> pGEX6P1 culture #2=(µl)
| + | |
| − | <br/> pRP261 culture #2=(µl)
| + | |
| − | <br/> pET303_aGFPnano_TEV_10His culture #1=6(µl)
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Test-digestion </strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion mixture 1:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit54">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">5-6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT54 TABLE [23274-23422] -->
| + | |
| − | <p> <strong>Digestion mixture 2:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit55">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">5-6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI-HF</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT55 TABLE [23451-23603] -->
| + | |
| − | <p> <strong>1% Agarose Gel of Test Digestion</strong> </p>
| + | |
| | | | |
| | + | .team_pic_div { |
| | + | font-size: 20px; |
| | + | text-align: justify; |
| | + | font-family: 'Open Sans', sans-serif; |
| | + | padding: 5% 10% 10% 10%; |
| | + | } |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/1/1c/T--Freiburg--CloningJournal18.png" ></center>
| + | .para_center_Quellen { |
| − |
| + | font-size: 15px; |
| − | </div>
| + | text-align: justify; |
| − | <!-- EDIT53 SECTION "28.07.16" [22117-23711] -->
| + | font-family: 'Arvo', serif; |
| − | <h3 class="sectionedit56"><a name="section290716" id="section290716">29.07.16</a></h3>
| + | padding: 5% 10% 5% 10%; |
| − | <div class="level3">
| + | } |
| − | <p> <strong>1) Mini-prep:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> A standard Qiagen Mini-Prep was performed with the following cultures:
| + | |
| − | <br/> pJET1.2-cotB cultures #2/3/4, pJET1.2-cotG cultures #1/2/3/4, pET303_aGFPnano_TEV_10His culture #1. </p>
| + | |
| − | <p> The DNA concentrations were measured with Nanodrop:
| + | |
| − | <br/> pJET1.2-cotB cultures #2=225,5(ng/µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #3=289,5(ng/µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #4=229,1(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #1=104,7(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #2=63,2(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #3=97,6(ng/µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #4=192,1(ng/µl)
| + | |
| − | <br/> pET303_aGFPnano_TEV_10His culture #1=135,1(ng/µl)
| + | |
| − | <br/> </p>
| + | |
| − | <p> µl taken for Test Digestion:
| + | |
| − | <br/> pJET1.2-cotB cultures #2=2(µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #3=2(µl)
| + | |
| − | <br/> pJET1.2-cotB cultures #4=2(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #1=5(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #2=6(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #3=5(µl)
| + | |
| − | <br/> pJET1.2-cotG cultures #4=3(µl)
| + | |
| − | <br/> pET303_aGFPnano_TEV_10His culture #1=4(µl)
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Test-digestion</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion mixture:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit57">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">5-6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI-HF</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer Smart-Cut</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT57 TABLE [24688-24846] -->
| + | |
| − | <p> <strong>1% Agerose Gel of Test Digestion</strong> </p>
| + | |
| − | <p>
| + | |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/fa/T--Freiburg--CloningJournal19.png" ></center>
| + | .para_center_without_margin_bottom { |
| | + | font-size: 20px; |
| | + | text-align: center; |
| | + | line-height: 120%; |
| | + | margin-left: 10%; |
| | + | margin-right: 100px; |
| | + | margin-bottom: 0px; |
| | + | font-family: 'Open Sans', sans-serif; |
| | + | } |
| | | | |
| − | </p>
| + | .para { |
| − | <p> After testdigestion pJET1.2-cotB#3 and pJET1.2-cotG#1 were sent to sequencing. </p>
| + | font-size: 10px; |
| − | </div>
| + | } |
| − | </div>
| + | |
| − | <!-- para20-->
| + | |
| − | </div> | + | |
| − | <!--color-->
| + | |
| − | <div class="color2">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT56 SECTION "29.07.16" [23712-25036] -->
| + | |
| − | <h3 class="sectionedit58"><a name="section300716" id="section300716">30.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Extension PCR</strong>
| + | |
| − | <br/> Extension PCR of of cgeA and aGFP-Nanobody to generate overhangs for Gibson cloning.
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit59">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Template</th>
| + | |
| − | <th class="col1">Primer</th>
| + | |
| − | <th class="col2">Annealing Temp. [°C]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pJET1.2_cgeA #2</td>
| + | |
| − | <td class="col1">oIG16_044 + 045</td>
| + | |
| − | <td class="col2">62</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1">oIG16_050 + 035</td>
| + | |
| − | <td class="col2">62</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pET303_aGFPnano_TEV_10His</td>
| + | |
| − | <td class="col1">oIG16_030 + 031</td>
| + | |
| − | <td class="col2">67</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> </td>
| + | |
| − | <td class="col1">oIG16_051 + 042</td>
| + | |
| − | <td class="col2">67</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT59 TABLE [25168-25337] -->
| + | |
| − | <p> *Reaction conditions* </p>
| + | |
| − | <div class="table sectionedit60">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">Volume[µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Primer fw</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer rv</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Q5 HiFi MasterMix</td>
| + | |
| − | <td class="col1">2x</td>
| + | |
| − | <td class="col2">25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultra pure H2O</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">19</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT60 TABLE [25361-25492] -->
| + | |
| − | <p> *PCR program*
| + | |
| − | <br/> Touchdown PCR </p>
| + | |
| − | <div class="table sectionedit61">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Step</th>
| + | |
| − | <th class="col1">Temperature [°C]</th>
| + | |
| − | <th class="col2">Duration</th>
| + | |
| − | <th class="col3">Repeats</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Initial denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2"> 5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">Annealing Temp + 10°C (-1°C per cycle) </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72</td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">10X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">Denaturation</td>
| + | |
| − | <td class="col1">98 </td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">Annealing</td>
| + | |
| − | <td class="col1">Annealing temp.</td>
| + | |
| − | <td class="col2">10 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">Elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">30 s</td>
| + | |
| − | <td class="col3">20X</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">Final elongation</td>
| + | |
| − | <td class="col1">72 </td>
| + | |
| − | <td class="col2">5 min</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">Storage</td>
| + | |
| − | <td class="col1">8</td>
| + | |
| − | <td class="col2"> - </td>
| + | |
| − | <td class="col3"> </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT61 TABLE [25525-25854] -->
| + | |
| − | <p> After PCR the samples were analyzed by agarose gel electrophoresis (1 % agarose TAE gel). </p>
| + | |
| − | <p>
| + | |
| | | | |
| | + | .level3 { |
| | + | font-size: 18px; |
| | + | } |
| | | | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/f1/T--Freiburg--CloningJournal20.png" ></center>
| + | .pinkpurple { |
| − |
| + | background-color: #871150; |
| | + | color: orange; |
| | + | } |
| | | | |
| | + | .level3 h3 { |
| | + | font-size: 36px; |
| | + | /*padding: 20px;*/ |
| | + | } |
| | | | |
| − | <strong>2) Gel extraction</strong>
| + | .para_20 { |
| − | <br/> The bands corresponding to the appropriate sizes were extracted using the QiaQuick gel extraction kit and the DNA was eluted in 30 µL of ultra pure water. The concentration was photometrically determined by a Nanodrop. </p>
| + | padding: 5% 15%; |
| − | <div class="table sectionedit62">
| + | } |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Sample</th>
| + | |
| − | <th class="col1">Concentration [ng/µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">cgeA_oIG16_044+045</td>
| + | |
| − | <td class="col1">85.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">cgeA_oIG16_50+35</td>
| + | |
| − | <td class="col1">62.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">aGFPnano_oIG16_31+30</td>
| + | |
| − | <td class="col1">93.7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">aGFPnano_oIG16_51+42</td>
| + | |
| − | <td class="col1">104</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT62 TABLE [26268-26405] -->
| + | |
| − | </div>
| + | |
| − | <!-- EDIT58 SECTION "30.07.16" [25037-26406] -->
| + | |
| − | <h3 class="sectionedit63"><a name="section310716" id="section310716">31.07.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Restriction digestion </strong> </p>
| + | |
| − | <p> Linearization of pSB1C3 for Gibson Cloning: </p>
| + | |
| − | <p> <strong>Digestion mixture</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit64">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">100ng/µL</td>
| + | |
| − | <td class="col2">5-6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT64 TABLE [26528-26678] -->
| + | |
| − | <p> <strong>2) Agarose Gel of pSB1c3</strong> </p>
| + | |
| − | <p>
| + | |
| − |
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/f6/T--Freiburg--CloningJournal21.png" ></center>
| + | |
| | | | |
| − | | + | </style> <head> <link href="https://fonts.googleapis.com/css?family=Open+Sans" rel="stylesheet"> <link href="https://fonts.googleapis.com/css?family=Sniglet" rel="stylesheet"> <link href="https://fonts.googleapis.com/css?family=Arvo" rel="stylesheet"> <link href="https://fonts.googleapis.com/css?family=Days+One" rel="stylesheet"> </head> </html> |
| − | </p>
| + | |
| − | <p> → followed by gel extraction for gibson assembly. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT63 SECTION "31.07.16" [26407-26831] -->
| + | |
| − | <h3 class="sectionedit65"><a name="section010816" id="section010816">01.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) NEBuilder® HiFi DNA Assembly Cloning</strong>: </p>
| + | |
| − | <p> Assembly of extensionPCR products into pSB1C3. </p>
| + | |
| − | <div class="table sectionedit66">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No.</th>
| + | |
| − | <th class="col1">amplified fragment1</th>
| + | |
| − | <th class="col2"> amplified fragment 2</th>
| + | |
| − | <th class="col3">digested Backbone</th>
| + | |
| − | <th class="col4" colspan="2">Resulting Plasmid</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1"> cgeA oIG16_50+35</td>
| + | |
| − | <td class="col2">aGFPnano_oIG16_51+42 </td>
| + | |
| − | <td class="col3"> XbaI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4"> pSB1C3_cgeA_aHelix_HA_aGFPnano pIG16_038</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1"> aGFPnano oIG16_30+47 </td>
| + | |
| − | <td class="col2 leftalign">cgeA oIG16_46+45 </td>
| + | |
| − | <td class="col3">EcoRI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4"> pSB1C3_aGFPnano_HA_aHelix_cgeA pIG16_039</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT66 TABLE [26947-27249] -->
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> DNA: 1.5 µL
| + | |
| − | <br/> Fragment1: 25 ng
| + | |
| − | <br/> Fragment2: 15 ng
| + | |
| − | <br/> linearized pSB1C3: 25 ng
| + | |
| − | <br/> 2X HiFi MasterMix: 5 µL
| + | |
| − | <br/> ultra pure water: 3.5 µL
| + | |
| − | <br/> </p>
| + | |
| − | <p> The reaction was incubated at 50 °C for 1 hour. 2 µL of the reaction mixture was transformed into chemically competent E.coli DH5alpha provided by the NEBuilder HiFi kit according to their protocol, plated on LB-agar plates containing chloramphenicol and incubated o/n at 37 °C.
| + | |
| − | <br/> The control reaction provided by the kit was performed according to the instructions of the manufacturer. </p>
| + | |
| − | <p> <strong>2) Digestion of pBS1C</strong>
| + | |
| − | <br/> <strong>Digestion mixture:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit67">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">148ng/µL</td>
| + | |
| − | <td class="col2">16</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">NEBuffer CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT67 TABLE [27859-27994] -->
| + | |
| − | <p> <strong>Agarose gel of pBS1c:</strong> </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/c/c9/T--Freiburg--CloningJournal22.png" ></center>
| + | |
| − | </p>
| + | |
| − | <p> → followed by a Gel extraction for Transformation of competent B.subtilis W168. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!--para20-->
| + | |
| − | </div>
| + | |
| − | <!--color-->
| + | |
| − | <div class="color3">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT65 SECTION "01.08.16" [26832-28174] -->
| + | |
| − | <h3 class="sectionedit68"><a name="section020816" id="section020816">02.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculations</strong>
| + | |
| − | <br/> Inoculation of 2 colonies from each plate with transformed E.coli containing pSB1C3_cgeA_aHelix_HA-Tag_aGFPnano and pSB1C3_aGFPnano_aHelix_HA-Tag_cgeA. Incubation o/n at 37 °C and 250 rpm.
| + | |
| − | <br/> Remaining E.coli from the transformation (see previous day) were plated on LB-agar plates supplemented with cml and incubated o/n at 37 °C. </p>
| + | |
| − | <p> <strong>2) Digestion of pBS1C (For transformation of B.subtilis)</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion mixture:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit69">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">700ng/µL</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">1.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">NEBuffer CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT69 TABLE [28640-28775] -->
| + | |
| − | <p>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/0c/T--Freiburg--CloningJournal23.png" ></center> → PCR purification was peformed </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT68 SECTION "02.08.16" [28175-28879] -->
| + | |
| − | <h3 class="sectionedit70"><a name="section030816" id="section030816">03.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> Plasmid preparation of the inoculated E.coli from the previous day using the QiaQuick Plasmid preparation kit according to the protocol of the manufacturer and eluted in 30 µL of ultra pure water. The DNA concentration was photometrically determined by a NanoDrop: </p>
| + | |
| − | <div class="table sectionedit71">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">sample</th>
| + | |
| − | <th class="col1">concentration [ng/µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano #1</td>
| + | |
| − | <td class="col1">69.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano #2</td>
| + | |
| − | <td class="col1">126.7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA #1</td>
| + | |
| − | <td class="col1">120.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA #2</td>
| + | |
| − | <td class="col1">182.1</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT71 TABLE [29184-29414] -->
| + | |
| − | <p> <strong>2) Testdigestion</strong>
| + | |
| − | <br/> 500 ng of DNA was treated with 5 unit of XbaI and PstI in 1x NEBuffer 2.1 in a total reaction volume of 20 µL. Incubation for 1hour at 37 °C. </p>
| + | |
| − | <p>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/3/3c/T--Freiburg--CloningJournal24.png" ></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) Inoculation</strong>
| + | |
| − | <br/> <strong>I</strong> Inoculation of E.coli DH5alpha containing an mCherry and GFP plasmid (provided by AG Weber) in 5 mL LB containig (Three colonies per plate were picked)
| + | |
| − | <br/> <strong>II</strong> E.coli DH5alpha containing plasmids sent from Poland (by Dr. Krystof Hinc) for the construction of fusion proteins for spore surface display arrived:
| + | |
| − | <br/> 1)pCotG-N
| + | |
| − | <br/> 2)pCotG-C
| + | |
| − | <br/> 3)pCotB-N
| + | |
| − | <br/> 4)pCotB-C
| + | |
| − | <br/> 5)pCgeA-C
| + | |
| − | <br/> 6)pCotZ-C
| + | |
| − | <br/> The E.coli were spread on LB-Agar plates containing ampicilin and incubated o/n at 37 °C.
| + | |
| − | <br/> <strong>III</strong>* 6 colonies per plate of E.coli DH5alpha containing pSB1C3_CgeA_alphaHelix_HA-Tag_aGFPnano or pSB1C3_aGFPnano_alphaHelix_HA-Tag_cgeA were inoculated in 5 mL LB-medium supplemented with chloramphenicol and incubated at 37 °C and 250rpm. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT70 SECTION "03.08.16" [28880-30417] -->
| + | |
| − | <h3 class="sectionedit72"><a name="section040816" id="section040816">04.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep pIG16_038 + pIG16_039, GFP + mCherry</strong>
| + | |
| − | <br/> Plasmid preparation of inoculated E.coli DH5alpha transformed with plasmids containing GFP and mCherry (from AG Weber) using the QiaQuick MiniPrep kit according to the instructions of the manufacturer. Elution with 30 µL of ultra pure water. The concentration was determined by Nanodrop. </p>
| + | |
| − | <div class="table sectionedit73">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No</th>
| + | |
| − | <th class="col1">Sample ID</th>
| + | |
| − | <th class="col2">Nucleic Acid Conc.</th>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1">h2o</td>
| + | |
| − | <td class="col2">0,9</td>
| + | |
| − | <td class="col3">ng/µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">pIG16_038 #1</td>
| + | |
| − | <td class="col2">106,9ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">pIG16_038 #2</td>
| + | |
| − | <td class="col2">92,4ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">pIG16_038 #3</td>
| + | |
| − | <td class="col2">77,3ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">5</td>
| + | |
| − | <td class="col1">pIG16_038 #4</td>
| + | |
| − | <td class="col2">64,4ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">6</td>
| + | |
| − | <td class="col1">pIG16_038 #5</td>
| + | |
| − | <td class="col2">63,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">7</td>
| + | |
| − | <td class="col1">pIG16_038 #6</td>
| + | |
| − | <td class="col2">55,4ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">8</td>
| + | |
| − | <td class="col1">pIG16_039 #1</td>
| + | |
| − | <td class="col2">105,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">9</td>
| + | |
| − | <td class="col1">pIG16_039 #2</td>
| + | |
| − | <td class="col2">101,3ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">10</td>
| + | |
| − | <td class="col1">pIG16_039 #3</td>
| + | |
| − | <td class="col2">73,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0">11</td>
| + | |
| − | <td class="col1">pIG16_039 #4</td>
| + | |
| − | <td class="col2">85,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row12">
| + | |
| − | <td class="col0">12</td>
| + | |
| − | <td class="col1">pIG16_039 #5</td>
| + | |
| − | <td class="col2">80,4ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row13">
| + | |
| − | <td class="col0">13</td>
| + | |
| − | <td class="col1">pIG16_039 #6</td>
| + | |
| − | <td class="col2">86,7ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row14">
| + | |
| − | <td class="col0">14</td>
| + | |
| − | <td class="col1">pIG16_024 GFP #1</td>
| + | |
| − | <td class="col2">93,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row15">
| + | |
| − | <td class="col0">15</td>
| + | |
| − | <td class="col1">pIG16_024 GFP #2</td>
| + | |
| − | <td class="col2">76,3ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row16">
| + | |
| − | <td class="col0">16</td>
| + | |
| − | <td class="col1">pIG16_024 GFP #3</td>
| + | |
| − | <td class="col2">100,9ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row17">
| + | |
| − | <td class="col0">17</td>
| + | |
| − | <td class="col1">pIG16_025mCherry#3</td>
| + | |
| − | <td class="col2">143,2ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row18">
| + | |
| − | <td class="col0">18</td>
| + | |
| − | <td class="col1">pIG16_025mCherry#2</td>
| + | |
| − | <td class="col2">175,8ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row19">
| + | |
| − | <td class="col0">19</td>
| + | |
| − | <td class="col1">pIG16_025mCherry#3</td>
| + | |
| − | <td class="col2">132,8ng/µl</td>
| + | |
| − | <td class="col3"></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT73 TABLE [30783-31386] -->
| + | |
| − | <p> <strong>2)Inoculation</strong>
| + | |
| − | <br/> Inoculation of the E.coli containing the plasmids sent from Poland. 4 Colonies from each plate were picked and inoculated in 5 mL of LB medium supplemented with Amp.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>3)ExtensionPCRs Q5 –> Gel+extraction</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit74">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> No. </th>
| + | |
| − | <th class="col1"> Template </th>
| + | |
| − | <th class="col2"> Primer: oIG16_# </th>
| + | |
| − | <th class="col3"> Annealing Temp[°C] </th>
| + | |
| − | <th class="col4"> Resulting construct </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1"> pET303_aGFPnano</td>
| + | |
| − | <td class="col2"> 030 + 047</td>
| + | |
| − | <td class="col3"> 67</td>
| + | |
| − | <td class="col4">Nano_HA_G4S</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1"> pET303_aGFPnano</td>
| + | |
| − | <td class="col2"> 053 + 042</td>
| + | |
| − | <td class="col3"> 67</td>
| + | |
| − | <td class="col4">G4S_HA_Nano</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1"> pJET_cotG</td>
| + | |
| − | <td class="col2"> 015 + 016</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4">HA_aHelix_CotG</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1"> pJET_cotG</td>
| + | |
| − | <td class="col2"> 011 + 013</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4">CotG_aHelix_HA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">5</td>
| + | |
| − | <td class="col1"> pSBBs4S_Sporovector</td>
| + | |
| − | <td class="col2"> 048 + 049</td>
| + | |
| − | <td class="col3"> 55</td>
| + | |
| − | <td class="col4">Bb-Prefix_PCotYZ_Bb-Suffix</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">6</td>
| + | |
| − | <td class="col1"> pJET_cgeA</td>
| + | |
| − | <td class="col2"> 046 + 045</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4">HA_G4S_CgeA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">7</td>
| + | |
| − | <td class="col1"> pJET_cgeA</td>
| + | |
| − | <td class="col2"> 050 + 052</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4">CgeA_G4S_HA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">8</td>
| + | |
| − | <td class="col1"> dH2O</td>
| + | |
| − | <td class="col2"> 046 + 045</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4">negative control</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">9</td>
| + | |
| − | <td class="col1"> dH2O</td>
| + | |
| − | <td class="col2"> 015 + 016</td>
| + | |
| − | <td class="col3"> 62</td>
| + | |
| − | <td class="col4"> negative control</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">10</td>
| + | |
| − | <td class="col1"> dH2O</td>
| + | |
| − | <td class="col2"> 030 + 047</td>
| + | |
| − | <td class="col3"> 67</td>
| + | |
| − | <td class="col4"> negative control</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT74 TABLE [31624-32170] -->
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit75">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">Volume µL</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">2XHiFi MasterMix</td>
| + | |
| − | <td class="col1">25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer1</td>
| + | |
| − | <td class="col1">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer2</td>
| + | |
| − | <td class="col1">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">template DNA</td>
| + | |
| − | <td class="col1">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">19.9</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT75 TABLE [32198-32313] -->
| + | |
| − | <p> <strong>PCR Program</strong>
| + | |
| − | <br/> Touchdown PCRs corresponding to the annealing temperatures.
| + | |
| − | <br/> </p>
| + | |
| − | <p> After amplification the samples were loaded on a 1% Agarose TAE gel. </p>
| + | |
| − | <p>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/e/e5/T--Freiburg--CloningJournal25.png" ></center>
| + | |
| − | </p>
| + | |
| − | <p> The bands corresponding to the expected fragment sizes were extracted from the gel, purified and eluted with 30 µL of ultra pure water. </p>
| + | |
| − | </div>
| + | |
| − | <!-- para-->
| + | |
| − | </div>
| + | |
| − | <!--color-->
| + | |
| − | </div>
| + | |
| − | <div class="color4">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT72 SECTION "04.08.16" [30418-32670] -->
| + | |
| − | <h3 class="sectionedit76"><a name="section050816" id="section050816">05.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Testdigestion</strong>
| + | |
| − | <br/> pIG16_038+039 were verified by testdigestion. 500 ng of DNA was treated with XbaI and PstI and analyzed by gel electrophoresis.
| + | |
| − | <br/>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/a9/T--Freiburg--CloningJournal26.png" ></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) MiniPrep</strong>
| + | |
| − | <br/> The inoculated cultures containing the plasmids sent from poland were preparated using the QiaQuick MiniPrep kit according to the protocol. The DNA was eluted with 30 µL of ultra pure water. The concentration was determined by Nanodrop.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>3) GFP purification</strong>
| + | |
| − | <br/> Sebastian from AG Weber gave a purified GFP (1.35 mg/mL) and mCherry (2 mg/mL).
| + | |
| − | <br/> Stored at 4 °C, protected from light. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT76 SECTION "05.08.16" [32671-33332] -->
| + | |
| − | <h3 class="sectionedit77"><a name="section060816" id="section060816">06.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) ExtensionPCR</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit78">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">template</th>
| + | |
| − | <th class="col1">Primer oIG16_</th>
| + | |
| − | <th class="col2">Annealing Temp. [°C]</th>
| + | |
| − | <th class="col3">Resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pJet1.2_cotG</td>
| + | |
| − | <td class="col1">011 + 013</td>
| + | |
| − | <td class="col2"> 62 </td>
| + | |
| − | <td class="col3">CotG_aHelix_HA</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT78 TABLE [33375-33486] -->
| + | |
| − | <p> After amplification the sample was loaded on a 1% Agarose TAE gel. The band corresponding to the expected size was extracted and purified.
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/83/T--Freiburg--CloningJournal27.png" ></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) 3A assembly</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion mixture PCotYZ:</strong>
| + | |
| − | <br/> The B.subtilis spore coat promoter PCotYZ was digested from the Sporovector pIG16_017 provided by Julia Bartels from the Mascher Lab at the TU Dresden. </p>
| + | |
| − | <div class="table sectionedit79">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">13.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">SpeI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer Cut Smart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT79 TABLE [33899-34055] -->
| + | |
| − | <p> <strong>Digestion mixture for pIG16_038+039:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit80">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">variable</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT80 TABLE [34100-34253] -->
| + | |
| − | <p> <strong>Digestion mixture for pBS1C:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit81">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2"> variable</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT81 TABLE [34290-34446] -->
| + | |
| − | <p> <strong>T4 Ligation</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Ligation mixture of plG16_38/39 #1:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit82">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C</td>
| + | |
| − | <td class="col1">2000ng/µL</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">plG_38</td>
| + | |
| − | <td class="col1">105ng/µL</td>
| + | |
| − | <td class="col2">4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">plG_39</td>
| + | |
| − | <td class="col1">104ng/µL</td>
| + | |
| − | <td class="col2">4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PcotYZ</td>
| + | |
| − | <td class="col1">24ng/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0" colspan="2">T-4 Ligase</td>
| + | |
| − | <td class="col2">1µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">NEBuffer T-4 Ligase</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT82 TABLE [34511-34712] -->
| + | |
| − | <p> The reaction was incubated for 30min at RT and transformed into chemically competent mix and go E. coli DH5alpha
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>3) Gibson assembly</strong>
| + | |
| − | <br/> Assembly of extensionPCR products into pSB1C3. </p>
| + | |
| − | <div class="table sectionedit83">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No.</th>
| + | |
| − | <th class="col1">amplified fragment1</th>
| + | |
| − | <th class="col2"> amplified fragment 2</th>
| + | |
| − | <th class="col3">digested Backbone</th>
| + | |
| − | <th class="col4" colspan="2">Resulting Plasmid</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1"> cgeA oIG16_050+052</td>
| + | |
| − | <td class="col2">aGFPnano_oIG16_53+42 </td>
| + | |
| − | <td class="col3"> XbaI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4"> pSB1C3_cgeA_G4S_HA_aGFPnano pIG16_040</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">aGFPnano oIG16_30+47</td>
| + | |
| − | <td class="col2">cotG oIG16_46+45</td>
| + | |
| − | <td class="col3">EcoRI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4"> pSB1C3_aGFPnano_HA_G4S_cgeA pIG16_041</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">aGFPnano oIG16_030+031</td>
| + | |
| − | <td class="col2">cotG oIG16_015+016</td>
| + | |
| − | <td class="col3">EcoRI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4"> pSB1C3_aGFPnano_HA_aHelix_cotG pIG16_042</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">cotG oIG16_011+013</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_051+042</td>
| + | |
| − | <td class="col3">EcoRI - pSB1C3 - SpeI</td>
| + | |
| − | <td class="col4">pSB1C3_cotG_aHelix_HA_aGFPnano pIG16_043</td>
| + | |
| − | <td class="col5"></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT83 TABLE [34902-35415] -->
| + | |
| − | <p>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> DNA: 1.5 µL
| + | |
| − | <br/> Fragment1: 25 ng
| + | |
| − | <br/> Fragment2: 15 ng
| + | |
| − | <br/> linearized pSB1C3: 25 ng
| + | |
| − | <br/> 2X HiFi MasterMix: 5 µL
| + | |
| − | <br/> ultra pure water: 3.5 µL
| + | |
| − | <br/> The reaction was incubated at 50 °C for 1 hour. 2µL of the reaction mix were transformed into chemically competent E.coli DH5alpha and spread on agarose LB plates supplemented with chloramphenicol. Incubation o/n at 37 °C.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>4) Testdigestion of GFP </strong>
| + | |
| − | <br/> For verification 500 ng of the plasmid was treated with XbaI, XhoI, BamHI in 1X NEBuffer 3.1 for 90 min at 37°C. The fragments were analyzed by agarose gel electrophoresis.
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/8a/T--Freiburg--CloningJournal28.png"></center>
| + | |
| − | | + | |
| − | amp;media=labor:2016_08_06_gfp_testdigestion_invert_label.png" class="mediacenter" alt="" width="300" /></a>
| + | |
| − | </p>
| + | |
| − | <p> <strong>5) Test Digestion Plasmids provided by Krystof Hinc</strong>
| + | |
| − | <br/> From university Gdansk. A set of vectors for surface display in B.subtilis. </p>
| + | |
| − | <p> <strong>Digestion mixture cgeA-C/cotZ-C:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit84">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">cgeA: #1/2/4=5 #3=8 cotZ: #1/2/3/4=7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT84 TABLE [36259-36441] -->
| + | |
| − | <p> <strong>Digestion mixture cotB-C/N:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit85">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">cotB-C: #1/2=5 #3/4=6 cotB-N #1=5 #2=6 #3/4=7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT85 TABLE [36477-36649] -->
| + | |
| − | <p> <strong>Digestion mixture cotG-C:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit86">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">#1/2/3/4=2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">BamHI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT86 TABLE [36683-36821] -->
| + | |
| − | <p> <strong>Digestion mixture cotG-N:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit87">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">#1/2/3=2 #4=6</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbhI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XhoI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer CutSmart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT87 TABLE [36855-37020] -->
| + | |
| − | <p> <strong>cgeA-C cotB-C/N</strong>
| + | |
| − |
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/fc/T--Freiburg--CloningJournal29.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>cotB-C/N</strong>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/b1/T--Freiburg--CloningJournal30.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>cot-Z</strong>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/aa/T--Freiburg--CloningJournal31.png"></center>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT77 SECTION "06.08.16" [33333-37274] -->
| + | |
| − | <h3 class="sectionedit88"><a name="section070816" id="section070816">07.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Repeat of 3A Assembly</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Ligation mixture of plG_38/39 #1:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit89">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C</td>
| + | |
| − | <td class="col1">2000ng/µL</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">plG16_038</td>
| + | |
| − | <td class="col1">105ng/µL</td>
| + | |
| − | <td class="col2">7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">plG16_039</td>
| + | |
| − | <td class="col1">104ng/µL</td>
| + | |
| − | <td class="col2">7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PcotYZ</td>
| + | |
| − | <td class="col1">24ng/µL</td>
| + | |
| − | <td class="col2">0.4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0" colspan="2">T4 Ligase</td>
| + | |
| − | <td class="col2">1µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">T4 Ligase reaction buffer</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT89 TABLE [37367-37579] -->
| + | |
| − | <p> <strong>2) Inoculation</strong>
| + | |
| − | <br/> Inoculation of Culture #1/2 with construct plG16_038 and Culture #1/2 with construct plG16_039. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="color5">
| + | |
| − | <div class="para_20">
| + | |
| − |
| + | |
| − | <h3 class="sectionedit90"><a name="section080816" id="section080816">08.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1)Miniprep</strong> </p>
| + | |
| − | <p> Standard Qiagen Miniprep of Culture #1/2 with construct plG16_38 and Culture #1/2 with construct plG16_39. </p>
| + | |
| − | <p> <strong>2) Digestion</strong> </p>
| + | |
| − | <p> <strong>Digestion mixture poG16_38/39:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit91">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1" colspan="2">500ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT91 TABLE [37898-38043] -->
| + | |
| − | <p> <strong>Digestion mixture pBS1C:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit92">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1" colspan="2">500ng/µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 2.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <p>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/0f/T--Freiburg--CloningJournal32.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>3) Repeat of 3A Assembly</strong> </p>
| + | |
| − | <p> <strong>Ligation mixture of plG_38/39 #1:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit93">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C</td>
| + | |
| − | <td class="col1">2000ng/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">plG_38</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">1.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">plG_39</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">1.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PcotYZ</td>
| + | |
| − | <td class="col1">24ng/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0" colspan="2">T-4 Ligase</td>
| + | |
| − | <td class="col2">1µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">NEBuffer T-4 Ligase</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT93 TABLE [38365-38572] -->
| + | |
| − | <p> <strong>4) Inoculation</strong>
| + | |
| − | <br/> Transformed E.coli containing the plasmids pIG16_040 - 043 were inoculated. 6 colonies per plate were picked and inoculated in 5 mL LB medium supplemented with chloramphenicol. Incubation o/n at 37 °C and 250 rpm. </p>
| + | |
| − | <p> <strong>5) Subcloning of PCotYZ</strong>
| + | |
| − | <br/> PCotYZ PCR product was subcloned into pJET2.1 vector according to protocol delivered with the kit. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT90 SECTION "08.08.16" [37699-38940] -->
| + | |
| − | <h3 class="sectionedit94"><a name="section090816" id="section090816">09.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> The inoculated E.coli containing the plasmids pIG16_040 - 043 were preparated using the QiaQuick MiniPrep kit. The DNA was eluted with 30 µL of ultra pure water. Verification of the plasmids was performed by testdigestion. 500 ng of DNA was treated with XbaI and PstI in 1X NEBuffer 3.1 for 90 min at 37 °C. </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/0a/T--Freiburg--CloningJournal33.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) Sequencing</strong>
| + | |
| − | <br/> pIG16_038#1 and pIG16_039#1 were sent to sequencing by GATCbiotech. </p>
| + | |
| − | <p> <strong>3) ExtensionPCRs</strong> </p>
| + | |
| − | <div class="table sectionedit95">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No.</th>
| + | |
| − | <th class="col1">Template</th>
| + | |
| − | <th class="col2">Primer oIG16_</th>
| + | |
| − | <th class="col3">Annealing temp [°C]</th>
| + | |
| − | <th class="col4">Resulting construct </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1">pGEX6P1</td>
| + | |
| − | <td class="col2">036 + 054</td>
| + | |
| − | <td class="col3">64</td>
| + | |
| − | <td class="col4">GST_HA_G4S</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">pGEX6P1</td>
| + | |
| − | <td class="col2">041 + 055</td>
| + | |
| − | <td class="col3">64</td>
| + | |
| − | <td class="col4">G4S_HA_GST</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">pJET_cotZ</td>
| + | |
| − | <td class="col2">006 + 008</td>
| + | |
| − | <td class="col3">58</td>
| + | |
| − | <td class="col4">HA_G4S_CotZ</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">pJET_cotZ</td>
| + | |
| − | <td class="col2">003 + 004</td>
| + | |
| − | <td class="col3">58</td>
| + | |
| − | <td class="col4">CotZ_G4S_HA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">5</td>
| + | |
| − | <td class="col1">pJET_cotG</td>
| + | |
| − | <td class="col2">014 + 016</td>
| + | |
| − | <td class="col3">62</td>
| + | |
| − | <td class="col4">HA_G4S_CotG</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">6</td>
| + | |
| − | <td class="col1">pJET_cotG</td>
| + | |
| − | <td class="col2">011 + 012</td>
| + | |
| − | <td class="col3">62</td>
| + | |
| − | <td class="col4">CotG_G4S_HA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">7</td>
| + | |
| − | <td class="col1">pJET_cotB</td>
| + | |
| − | <td class="col2">022 + 024</td>
| + | |
| − | <td class="col3">56</td>
| + | |
| − | <td class="col4">HA_G4S_CotB</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">8</td>
| + | |
| − | <td class="col1">pJET_cotB</td>
| + | |
| − | <td class="col2">019 + 020</td>
| + | |
| − | <td class="col3">56</td>
| + | |
| − | <td class="col4">CotB_G4S_HA</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT95 TABLE [39486-39862] -->
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit96">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">2X Q5 MasterMix</td>
| + | |
| − | <td class="col1">25 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer1</td>
| + | |
| − | <td class="col1">2.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer2</td>
| + | |
| − | <td class="col1">2.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Template</td>
| + | |
| − | <td class="col1">0.1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">water</td>
| + | |
| − | <td class="col1">19.9 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT96 TABLE [39890-40005] -->
| + | |
| − | <p> <strong>PCR Program</strong>
| + | |
| − | <br/> Touchdown PCRs corresponding to the respective annealing temperatures. </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/2/2a/T--Freiburg--CloningJournal34.png"></center>
| + | |
| − | | + | |
| − | </p>
| + | |
| − | <p> <strong>3) Inoculation of pJET-PcotYZ colonies</strong> </p>
| + | |
| − | <p> Tree colonies from the the plate 1 (Wlad) and Tree colonies from the the plate 2 (iGEM) were picked and inoculated. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT94 SECTION "09.08.16" [38941-40322] -->
| + | |
| − | <h3 class="sectionedit97"><a name="section100816" id="section100816">10.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Gelextraction of ExtensionPCRs</strong> Was performed according to the protocol of the manufacturer. The samples were extracted from the gel from the previous day. </p>
| + | |
| − | <p> <strong>2) Sequence analysis</strong>
| + | |
| − | <br/> pIG16_038 + 039 #1
| + | |
| − | <br/> Sequencing showed that the samples were switched at some point. pIG16_039 contained an additional insert of ~9 bp between the alpha helical linker and the HA-tag. New samples have to be prepared for analysis. </p>
| + | |
| − | <p> <strong>3) Reinoculation </strong>
| + | |
| − | <br/> New colonies containing pIG16_038#2 039#2 were inoculated in 5 mL of LB medium supplemented with chloramphenicol. </p>
| + | |
| − | <p> <strong>4) Miniprep of pJET-PCotYZ</strong> </p>
| + | |
| − | <p> Standard Qiagen miniprep was performed with 3 cultures from the the plate 1 and 3 cultures from the the plate 2 . </p>
| + | |
| − | <p> <strong>5) Test digestion pJET-PcotYZ</strong> </p>
| + | |
| − | <p> <strong>Digestion pJET-PcotYZ:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit98">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">500ng/µL</td>
| + | |
| − | <td class="col2">1-3</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">SpeI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer Cut Smart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT98 TABLE [41117-41272] -->
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/e/e0/T--Freiburg--CloningJournal35.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> Culture #1 was sent to sequencing. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT97 SECTION "10.08.16" [40323-41376] -->
| + | |
| − | <h3 class="sectionedit99"><a name="section110816" id="section110816">11.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> Plasmid preparation of pIG16_038#2 and pIG16_039#2 using the QiaQuick MiniPrep kit. The DNA was eluted with 30 µL of ultra pure water.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>1) Sequencing</strong> The plasmids pIG16_038#2, pIG16_039#2, pIG16_040#2, pIG16_041#3, pIG16_IG16_042#3 and pJET1.2-PCotYZ#1 were sent to sequencing by GATCbiotech. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT99 SECTION "11.08.16" [41377-41719] -->
| + | |
| − | <h3 class="sectionedit100"><a name="section120816" id="section120816">12.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Sequencing Results</strong>
| + | |
| − | <br/> pIG16_040 - pIG16_042 could be confirmed by sequencing. Glycerol stock was prepared.
| + | |
| − | <br/> The samples from pIG16_038 and pIG16_039 were switched in previous steps. –> switched back. pIG16_039 was confirmed by sequencing.
| + | |
| − | <br/> pIG16_038 had a 1bp deletion. Further colonies pIG16_038#3 & #4 were sent to sequencing by GATC-Biotech.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2)MiniPrep of pIG16_039-042 and PcotYZ</strong>
| + | |
| − | <br/> Standard Quiagen Miniprep was performed. </p>
| + | |
| − | <p> <strong>3) Digestion of pIG16_039-042, PcotYZ</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion mixture pIG16_39-42:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit101">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">2500ng</td>
| + | |
| − | <td class="col2">5µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer 3.1</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT101 TABLE [42271-42417] -->
| + | |
| − | <p> <strong>Digestion mixture PcotYZ:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit102">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">DNA</td>
| + | |
| − | <td class="col1">5000ng</td>
| + | |
| − | <td class="col2">10</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">EcoRI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">SpeI</td>
| + | |
| − | <td class="col1">20u/µL</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">NEBuffer Cut Smart</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 50 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT102 TABLE [42451-42598] -->
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/b1/T--Freiburg--CloningJournal36.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>4) 3A Assembly</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Ligation mixture of plG_38/39 #1:</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit103">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume [µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C</td>
| + | |
| − | <td class="col1">50ng</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">plG_39-41</td>
| + | |
| − | <td class="col1">20ng</td>
| + | |
| − | <td class="col2">1</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">plG_42</td>
| + | |
| − | <td class="col1">25ng/µL</td>
| + | |
| − | <td class="col2">11</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PcotYZ</td>
| + | |
| − | <td class="col1">5.7ng</td>
| + | |
| − | <td class="col2">0.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">T-4 Ligase</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">1µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">NEBuffer T-4 Ligase</td>
| + | |
| − | <td class="col1">10X</td>
| + | |
| − | <td class="col2">2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">ultra pure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">ad 20 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT103 TABLE [42730-42927] -->
| + | |
| − | <p> <strong>5) Gibson Cloning</strong>
| + | |
| − | <br/> <strong>1.33x MasterMix preparation.</strong>
| + | |
| − | <br/> 5X Iso buffer: 50 µL
| + | |
| − | <br/> T5 Exonuclease (NEB): 1 µL (Diluted 1:10 in ultra pure water)
| + | |
| − | <br/> Taq Ligase (NEB): 25 µL
| + | |
| − | <br/> Phusion Polymerase (NEB): 3.125 µL
| + | |
| − | <br/> Nuclease free water: 108.4 µL
| + | |
| − | <br/> Aliquots: 7.5 µL, stored at -20 °C </p>
| + | |
| − | <p> <strong>Assemblies</strong>
| + | |
| − | <br/> All samples were adjusted to 50 ng/µL </p>
| + | |
| − | <div class="table sectionedit104">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No.</th>
| + | |
| − | <th class="col1">Fragment 1</th>
| + | |
| − | <th class="col2">Fragment 2</th>
| + | |
| − | <th class="col3">digested backbone</th>
| + | |
| − | <th class="col4">resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1">GST oIG16_036+054</td>
| + | |
| − | <td class="col2">CotG oIG16_014+016</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_046</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">GST oIG16_036+054</td>
| + | |
| − | <td class="col2">CgeA oIG16_046+045</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_047</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">GST oIG16_036+054</td>
| + | |
| − | <td class="col2">CotZ 006+008</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_048</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">GST oIG16_036+054</td>
| + | |
| − | <td class="col2">CotB 022+024</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_049</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">5</td>
| + | |
| − | <td class="col1">GST oIG16_041+055</td>
| + | |
| − | <td class="col2">CgeA 050+052</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_050</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">6</td>
| + | |
| − | <td class="col1">GST oIG16_041+055</td>
| + | |
| − | <td class="col2">CotZ 003+004</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_051</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">7</td>
| + | |
| − | <td class="col1">GST oIG16_041+055</td>
| + | |
| − | <td class="col2">CotB 019+020</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_052</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">8</td>
| + | |
| − | <td class="col1">aGFPnano oIG16_030+047</td>
| + | |
| − | <td class="col2">CotG 014+016</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_053</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">9</td>
| + | |
| − | <td class="col1">aGFPnano oIG16_030+047</td>
| + | |
| − | <td class="col2">CotZ 006+008</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_054</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">10</td>
| + | |
| − | <td class="col1">aGFPnano oIG16_030+047</td>
| + | |
| − | <td class="col2">CotB 022+024</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_055</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0">11</td>
| + | |
| − | <td class="col1">positive control</td>
| + | |
| − | <td class="col2">positive control</td>
| + | |
| − | <td class="col3">postive control</td>
| + | |
| − | <td class="col4">positive control from NEB HiFi Assembly kit</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT104 TABLE [43269-43981] -->
| + | |
| − | <p> 0.5 µL of each fragment and 0.5 µL of the digested backbone were mixed with 1µL of ultra pure water and added to the 1.33x Gibson Mix and incubated for 60 min at 50 °C. 5µL of the reaction mix were used to transform chemically competent E.coli DH5alpha. Subsequently, 300 µL of LB medium were added. The Bacteria were incubated at 37 °C and 250 rpm and spread on Agar-LB plates supplemented with chloramphenicol. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!-- para-->
| + | |
| − | </div>
| + | |
| − | <!-- class-->
| + | |
| − | <div class="color6">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT100 SECTION "12.08.16" [41720-44406] -->
| + | |
| − | <h3 class="sectionedit105"><a name="section130816" id="section130816">13.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Extension PCR</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit106">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">template</th>
| + | |
| − | <th class="col1">Primer</th>
| + | |
| − | <th class="col2">Resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pJET1.2_CotZ</td>
| + | |
| − | <td class="col1">oIG16_007+008</td>
| + | |
| − | <td class="col2">HA_alphaHelix_CotZ_pSB1C3-OH</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pJET1.2_CotZ</td>
| + | |
| − | <td class="col1">oIG16_003+005</td>
| + | |
| − | <td class="col2">pSB1C3-OH_CotZ_alphaHelix_HA</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT106 TABLE [44449-44602] -->
| + | |
| − | <p> <strong>Reaction conditions</strong> </p>
| + | |
| − | <div class="table sectionedit107">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">volume</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">2XMM</td>
| + | |
| − | <td class="col1">25 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer1</td>
| + | |
| − | <td class="col1">2.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer2</td>
| + | |
| − | <td class="col1">2.5 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">template</td>
| + | |
| − | <td class="col1">0.1 µL</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">dH2O</td>
| + | |
| − | <td class="col1">19.9 µL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT107 TABLE [44628-44731] -->
| + | |
| − | <p> <strong>Cycler Program</strong> Touchdown58 </p>
| + | |
| − | <p> The samples analyzed by agarose gel electrophoresis and the bands corresponding to the expected sizes were extracted and purified using the Qiagen gel extraction kit. </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/fe/T--Freiburg--CloningJournal37.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) Inoculation</strong> The transformed E.coli from the Gibson assembly (previous day) were inoculated in 5 mL LB-medium supplemented with chloramphenicol and incubated o/n at 37 °C and 250 rpm. </p>
| + | |
| − | <p> <strong>3) Gibson assembly</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit108">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">No.</th>
| + | |
| − | <th class="col1">Fragment1</th>
| + | |
| − | <th class="col2">Fragment2</th>
| + | |
| − | <th class="col3">Digested Backbone</th>
| + | |
| − | <th class="col4">resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1">pJet1.2_cotZ oIG16_007+008</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_30+31</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_056</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">pJet1.2_cotZ oIG16_006+008</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_30+47</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_057</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">pJet1.2_cotZ oIG16_003+005</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_51+42</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_058</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">pJet1.2_cotZ oIG16_003+004</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_53+42</td>
| + | |
| − | <td class="col3">pSB1C3</td>
| + | |
| − | <td class="col4">pIG16_059</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">5</td>
| + | |
| − | <td class="col1">positive control</td>
| + | |
| − | <td class="col2">positive control</td>
| + | |
| − | <td class="col3">positive control</td>
| + | |
| − | <td class="col4">C+ from NEB</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT108 TABLE [45212-45618] -->
| + | |
| − | <p> 0.5 µL from each fragment were mixed with 1 µL of ultra pure water, added to 1.33x Gibson Master Mix and incubated at 50 °C for 1 h. 2µL of the reaction mix were transformed into chemically competent E.coli DH5alpha. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT105 SECTION "13.08.16" [44407-45842] -->
| + | |
| − | <h3 class="sectionedit109"><a name="section140816" id="section140816">14.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) MiniPrep</strong>
| + | |
| − | <br/> Plasmid preparation of pIG16_046 - 055 using the QiaQuick MiniPrep kit. The DNA was eluted in 30 µL of ultra pure water. Verification of the plasmids by Testdigestion using. 500 ng of DNA was treated with 2 unit of XbaI and PstI in 1X NEBuffer 3.1 for 1 hour. . Unexpected band patterns. The DNA was not completly digested. </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/7/7a/T--Freiburg--CloningJournal38.png"></center>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/0a/T--Freiburg--CloningJournal39.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> <strong>2) Inoculation</strong>
| + | |
| − | <br/> The transformed E.coli with the Gibson assemblies from the previous day were inoculated in 5 mL LB-medium supplemented with chloramphenicol </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT109 SECTION "14.08.16" [45843-46508] -->
| + | |
| − | <h3 class="sectionedit110"><a name="section150816" id="section150816">15.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Testdigestion</strong>
| + | |
| − | <br/> The digestion of pIG16_046 - 055 from the previous day was repeated. 500 ng of DNA was treated with 4 unit of XbaI and PstI in 1X NEBuffer 2.1 for 90 min at 37 °C. </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/c/c7/T--Freiburg--CloningJournal40.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> Positive samples were sent to sequencing:
| + | |
| − | <br/> 1) pIG16_047#3
| + | |
| − | <br/> 2) pIG16_048#1
| + | |
| − | <br/> 3) pIG16_050#5
| + | |
| − | <br/> 4) pIG16_051#1
| + | |
| − | <br/> 5) pIG16_054#1
| + | |
| − | <br/> 6) pIG16_055#2
| + | |
| − | <br/> </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT110 SECTION "15.08.16" [46509-46935] -->
| + | |
| − | <h3 class="sectionedit111"><a name="section160816" id="section160816">16.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Production of competent DH5-alpha E. coli</strong>
| + | |
| − | <br/> E. colis were made competent according to zymo research Mix and go protocol </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT111 SECTION "16.08.16" [46936-47082] -->
| + | |
| − | <h3 class="sectionedit112"><a name="section170816" id="section170816">17.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Inoculation</strong>
| + | |
| − | <br/> Since pIG16_046, 049, 052 were all negative 5 additional colonies were picked and inoculated in 5 mL of LB medium supplemented with chloramphenicol.
| + | |
| − | <br/> 5 colonies of the E.coli transformed with the 3A assembly reaction (pIG16_062 - 065) were pickend and inoculated in 5 mL of LB medium supplemented with ampicilin.
| + | |
| − | <br/> 5 colonies of the E.coli transformed with the Gibson assembly reaction (pIG16_057 - 059).
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Glycerol stocks</strong>
| + | |
| − | <br/> Preparation of Glycerol stocks of pIG16_038, 039, 040, 041, 042 </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT112 SECTION "17.08.16" [47083-47624] -->
| + | |
| − | <h3 class="sectionedit113"><a name="section180816" id="section180816">18.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Plasmid preparation </strong>
| + | |
| − | <br/> Preparation of plasmids from transformed E.coli containing the constructs pIG16_46 + 049 + 052+ 058+ 059+ 062+ 063+ 064+ 065 </p>
| + | |
| − | <p> <strong>2) Testdigestion</strong>
| + | |
| − | <br/> 3A assembly: NotI + XhoI
| + | |
| − | <br/> Gibson assembly: XbaI + PstI
| + | |
| − | <br/> </p>
| + | |
| − | <p>
| + | |
| − | | + | |
| − | <center><img class="something" src="https://static.igem.org/mediawiki/2016/c/cb/T--Freiburg--CloningJournal41.png"></center>
| + | |
| − | </p>
| + | |
| − | <p> Digestions partially incomplete.
| + | |
| − | <br/> Samples sent to sequencing: </p>
| + | |
| − | <p> <strong>3) Sequencing</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit114">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Sample</th>
| + | |
| − | <th class="col1">Sequencing Primer</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pIG16_046#7</td>
| + | |
| − | <td class="col1">oIG16_039</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pIG16_046#7</td>
| + | |
| − | <td class="col1">oIG16_040</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pIG16_049#6</td>
| + | |
| − | <td class="col1">oIG16_039</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">pIG16_049#6</td>
| + | |
| − | <td class="col1">oIG16_040</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">pIG16_052#10</td>
| + | |
| − | <td class="col1">oIG16_039</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">pIG16_052#10</td>
| + | |
| − | <td class="col1">oIG16_040</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row7">
| + | |
| − | <td class="col0">pIG16_058#2</td>
| + | |
| − | <td class="col1">oIG16_039</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row8">
| + | |
| − | <td class="col0">pIG16_058#2</td>
| + | |
| − | <td class="col1">oIG16_040</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row9">
| + | |
| − | <td class="col0">pIG16_059#5</td>
| + | |
| − | <td class="col1">oIG16_039</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row10">
| + | |
| − | <td class="col0">pIG16_059#5</td>
| + | |
| − | <td class="col1">oIG16_040</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row11">
| + | |
| − | <td class="col0">pIG16_062#3</td>
| + | |
| − | <td class="col1">oIG16_028</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row12">
| + | |
| − | <td class="col0">pIG16_062#3</td>
| + | |
| − | <td class="col1">oIG16_029</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row13">
| + | |
| − | <td class="col0">pIG16_063#2</td>
| + | |
| − | <td class="col1">oIG16_028</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row14">
| + | |
| − | <td class="col0">pIG16_063#2</td>
| + | |
| − | <td class="col1">oIG16_029</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row15">
| + | |
| − | <td class="col0">pIG16_064#3</td>
| + | |
| − | <td class="col1">oIG16_028</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row16">
| + | |
| − | <td class="col0">pIG16_064#3</td>
| + | |
| − | <td class="col1">oIG16_029</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row17">
| + | |
| − | <td class="col0">pIG16_065#5</td>
| + | |
| − | <td class="col1">oIG16_028</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row18">
| + | |
| − | <td class="col0">pIG16_065#5</td>
| + | |
| − | <td class="col1">oIG16_029</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT114 TABLE [48062-48522] -->
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <!-- para20-->
| + | |
| − | </div>
| + | |
| − | <!-- color -->
| + | |
| − | <div class="color7">
| + | |
| − | <div class="para_20">
| + | |
| − | <!-- EDIT113 SECTION "18.08.16" [47625-48524] -->
| + | |
| − | <h3 class="sectionedit115"><a name="section190816" id="section190816">19.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong>1) Glycerol stock </strong>
| + | |
| − | <br/> Preparation of a 15 % Glycerol stock of pIG16_039 and storage at - 80 °C. </p>
| + | |
| − | <p> <strong>2) Reinoculation </strong>
| + | |
| − | <br/> New 6 mL LB cultures were prepared for pIG16_047#3, 050#5, 048#4, 051#1 054#4 and incubated o/n at 37 °C and 250 rpm. For subsequent 3A assembly. </p>
| + | |
| − | <p> <strong>3) Preparation of competent E.coli</strong> </p>
| + | |
| − | <p> The Ecoli strain DH5-alpha was prepered and made competent according to Zymoreserch MIX and GO kit. </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT115 SECTION "19.08.16" [48525-48960] -->
| + | |
| − | <h3 class="sectionedit116"><a name="section200816" id="section200816">20.08.16</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p> <strong> 1) Extension PCR</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit117">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Template</th>
| + | |
| − | <th class="col1">Primer</th>
| + | |
| − | <th class="col2">Resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pJet1.2_CotG</td>
| + | |
| − | <td class="col1">oIG056+012</td>
| + | |
| − | <td class="col2">pSB1C3-OH_CotG_G4S_HA</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pJet1.2_CotG</td>
| + | |
| − | <td class="col1">oIG16_056+013</td>
| + | |
| − | <td class="col2">pSB1C3-OH_CotG_aHelix_HA</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT117 TABLE [49006-49145] -->
| + | |
| − | <p> <strong>Reaction conditions</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit118">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume[µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">Q5 MasterMix</td>
| + | |
| − | <td class="col1">2X</td>
| + | |
| − | <td class="col2">25</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">Primer1</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">Primer2</td>
| + | |
| − | <td class="col1">10 µM</td>
| + | |
| − | <td class="col2">2.5</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">ultrapure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">20.0</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">template DNA</td>
| + | |
| − | <td class="col1"> </td>
| + | |
| − | <td class="col2">0.1</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT118 TABLE [49173-49319] -->
| + | |
| − | <p> <strong>Thermal Cycling</strong>
| + | |
| − | <br/> Touchdown 62
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>2) Gibson Assembly </strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit119">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">Number</th>
| + | |
| − | <th class="col1">Fragment 1</th>
| + | |
| − | <th class="col2">Fragment 2</th>
| + | |
| − | <th class="col3">Linearized backbone</th>
| + | |
| − | <th class="col4">Resulting construct</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">1</td>
| + | |
| − | <td class="col1">CotG oIG16_056+013</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_051+042</td>
| + | |
| − | <td class="col3">pSB1C3 XbaI+SpeI</td>
| + | |
| − | <td class="col4">pIG16_043</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2</td>
| + | |
| − | <td class="col1">CotG oIG16_056+012</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_053+042</td>
| + | |
| − | <td class="col3">pSB1C3 XbaI+SpeI</td>
| + | |
| − | <td class="col4">pIG16_065</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">3</td>
| + | |
| − | <td class="col1">CotG oIG16_056+013</td>
| + | |
| − | <td class="col2">GST oIG16_041+055</td>
| + | |
| − | <td class="col3">pSB1C3 XbaI+SpeI</td>
| + | |
| − | <td class="col4">pIG16_066</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">4</td>
| + | |
| − | <td class="col1">CotG oIG16_014+016</td>
| + | |
| − | <td class="col2">aGFPnano oIG16_030+047</td>
| + | |
| − | <td class="col3">pSB1C3 XbaI+SpeI</td>
| + | |
| − | <td class="col4">pIG16_053</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT119 TABLE [49387-49744] -->
| + | |
| − | <p> The concentration of all fragments was adjusted to 50 ng/µL. 0.5 µL of each fragment were mixed alongside with 0.5 µL of the linearized vector and added to the 1.33x Gibson MasterMix and incubated for 60 min at 50 °C.
| + | |
| − | <br/> 5µL of the Gibson mix were transformed into chemically competent E.coli DH5alpha and spread on LB agar plate supplemented with chloramphenicol.
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong> 3) Digestion for 3 A Assembly </strong>
| + | |
| − | <br/> </p>
| + | |
| − | <p> <strong>Digestion of PcotYZ</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit120">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume[µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">PcotYZ</td>
| + | |
| − | <td class="col1">6000ng</td>
| + | |
| − | <td class="col2">20µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">CS Buffer</td>
| + | |
| − | <td class="col1">10x</td>
| + | |
| − | <td class="col2">5µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI-HF</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">1.2µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">SpeI-HF</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">1.2µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultrapure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">50.0</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT120 TABLE [50185-50341] -->
| + | |
| − | <p> <strong>Digestion of pBS1C and pBS4S</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit121">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume[µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pBS1C and pBS4S</td>
| + | |
| − | <td class="col1">5000ng</td>
| + | |
| − | <td class="col2">2,5µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">2.1 Buffer</td>
| + | |
| − | <td class="col1">10x</td>
| + | |
| − | <td class="col2">5µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">EcoRI-HF</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">1.2µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">1.2µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultrapure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">50.0</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT121 TABLE [50378-50542] -->
| + | |
| − | <p> <strong>Digestion of pIG16_38 47 48 50 51 54</strong>
| + | |
| − | <br/> </p>
| + | |
| − | <div class="table sectionedit122">
| + | |
| − | <table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">component</th>
| + | |
| − | <th class="col1">concentration</th>
| + | |
| − | <th class="col2">volume[µL]</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pIG16_#</td>
| + | |
| − | <td class="col1">3000ng</td>
| + | |
| − | <td class="col2">20µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">3.1 Buffer</td>
| + | |
| − | <td class="col1">10x</td>
| + | |
| − | <td class="col2">5µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">XbaI</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">0.6µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">PstI</td>
| + | |
| − | <td class="col1">20u/µl</td>
| + | |
| − | <td class="col2">0.6µl</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">ultrapure water</td>
| + | |
| − | <td class="col1">-</td>
| + | |
| − | <td class="col2">50.0</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT122 TABLE [50588-50739] -->
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | </body>
| + | |
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