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| − | <a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><h2><B>Microbiology Notebook</B></h2><a/>
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| − | </div>
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| − |
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| − | <div id="home">
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| − | <center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center>
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| − | </div>
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| − | <div id="week15">
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| − | <p><h5><B>Week 4</B></h5></p>
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| − | <p><h3><B>September 12, 2016:</B></h3></p>
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| − | <p>
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| − | <a href="#exp1"><h4> Digestion of the plasmid pSB1C3 </a></br>
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| − | </p>
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| − | <p><h3><B>September 15, 2016:</B></h3></p>
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| − | <p>
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| − | <a href="#exp6"><h4> Measure the amount of DNA extracted from the miniprep </h4></a></br>
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| − |
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| − | <a href="#exp7"><h4> Digestion of the plasmid pSB1C3 and inserts A1/A2/B1/B2/C1/C2/D1/D2/E1/E2 </h4></a></br>
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| − | <a href="#exp8"><h4>Electrophoresis on agarose gel</h4></a></br>
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| − | <a href="#exp9"><h4>DNA extraction</h4></a></br>
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| − | </p>
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| − | <p><h3><B>September 16, 2016:</B></h3></p>
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| − | <p>
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| − | <a href="#exp10"><h4> Measure the amount of DNA extracted from the miniprep </h4></a></br>
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| − | </p>
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| − |
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| − | <div class="lightbox" id="exp1">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption>
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| − | <p>
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| − | <U> Aim:</U> To have the right restriction sites to perform the ligation and the cloning.</br>
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| − | We choose appropriate restriction sites based on our inserts.</br></br>
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| − |
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| − | <U> Protocol:</U> follow in this link</br></br>
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| − | <U>What we did in the lab:</U></br>
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| − | <U>Materials:</U></br>
| |
| − | • Restriction enzymes: XbaI, SpeI (New England Biolabs, NEB)</br>
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| − | • Restriction enzyme buffers </br>
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| − | • 37°C water bath</br>
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| − | • UV spectrophotometer</br>
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| − | • pSB1C3 (48ng/µL)</br></br>
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| − |
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| − |
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| − | <U>Method:</U></br>
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| − | Mix all the reagents and let digest during 1 hr at 37°C </br></br>
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| − | Big volumes must be added first!</br></br>
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| − |
| |
| − | <table>
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| − | <thead>
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| − | <tr>
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| − | <th>Reactants</th>
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| − | <th>pSB1C3</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>Vol<sub>DNA</sub></p></strong></td>
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| − | <td>25 µL </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>Vol<sub>XbaI</sub></p></strong></td>
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| − | <td>10 µL </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>Vol<sub>SpeI</sub></p></strong></td>
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| − | <td>10 µL </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>Vol<sub> buffer (10X) (Cutsmart) </sub></p></strong></td>
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| − | <td>5 µL </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>Vol<sub>total</sub></p></strong></td>
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| − | <td>50 µL </td>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | <center>Volumes</center></br></br></br>
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| − |
| |
| − | 2. Incubate 10 min at 65°C to inactivate the enzymes.</br>
| |
| − | 3. Add 2 µL of rSAP in order to perform the dephosphorylation.</br>
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| − | 3. Store at -20°C</br></br>
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| − |
| |
| − | </p>
| |
| − | </figcaption>
| |
| − | </figure>
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| − | </div>
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| − |
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| − |
| |
| − | <div class="lightbox" id="exp2">
| |
| − | <figure>
| |
| − | <a href="#" class="closemsg"></a>
| |
| − | <figcaption>
| |
| − | <p>
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| − | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher)</br></br>
| |
| − |
| |
| − | <U>What we did in the lab:</U></br>
| |
| − | <U>Materials:</U></br>
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| − | • Nanodrop (Thermofisher)</br>
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| − | • Elution buffer from QIAGEN kit</br>
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| − | • Microbiology equipment (Follow this link)</br></br>
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| − |
| |
| − | <U>Method:</U></br>
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| − | Analyze absorbance at 260nm</br>
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| − | Clean the Nanodrop with water</br>
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| − | Make the blank with 1ul of elution buffer</br>
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| − | Put 1ul of your sample on the Nanodrop</br>
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| − | Make the measure and clean the Nanodrop between each measure</br></br>
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| − |
| |
| − | <U>Results:</U></br>
| |
| − | <table>
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Absorbance at 260nm</th>
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| − | <th>A260/280</th>
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| − | <th>Concentration (ng/µL )</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>pSB1C3 (1)</p></strong></td>
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| − | <td>1.94</td>
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| − | <td>115.7</td>
| |
| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>pSB1C3 (2)</p></strong></td>
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| − | <td>1.93</td>
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| − | <td>13.0</td>
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| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>B1-col6</p></strong></td>
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| − | <td>1.88</td>
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| − | <td>393.3</td>
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| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>B1-col8<sub>(diluted 1/2)</sub></p></strong></td>
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| − | <td>1.93</td>
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| − | <td>74.1</td>
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| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>B2-col7</p></strong></td>
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| − | <td>1.89 </td>
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| − | <td>84.8</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B2-col9</p></strong></td>
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| − | <td>1.90 </td>
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| − | <td>307.1</td>
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| − | </tr>
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| − | </tbody>
| |
| − | </table>
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| − | <center>Absorbance</center></br></br></br>
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| − |
| |
| − | </p>
| |
| − | </figcaption>
| |
| − | </figure>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="lightbox" id="exp7">
| |
| − | <figure>
| |
| − | <a href="#" class="closemsg"></a>
| |
| − | <figcaption>
| |
| − | <p>
| |
| − | <U> Aim:</U> To have the right restriction sites to perform the ligation and the cloning.</br>
| |
| − | We choose appropriate restriction sites based on our inserts.</br></br>
| |
| − |
| |
| − | <U> Protocol:</U> follow in this link</br></br>
| |
| − | <U>What we did in the lab:</U></br>
| |
| − | <U>Materials:</U></br>
| |
| − | • Restriction enzymes: XbaI, SpeI (New England Biolabs, NEB)</br>
| |
| − | • Restriction enzyme buffers </br>
| |
| − | • 37°C water bath</br>
| |
| − | • UV spectrophotometer</br>
| |
| − | • pSB1C3 (48ng/µL)</br></br>
| |
| − |
| |
| − | <U>Method:</U></br>
| |
| − | Mix all the reagents and let digest during 1 hr at 37°C </br></br>
| |
| − | Big volumes must be added first!</br></br>
| |
| − |
| |
| − |
| |
| − | <table>
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Reactants</th>
| |
| − | <th>Each sample</th>
| |
| − | <th>Global mix</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>DNA</sub></p></strong></td>
| |
| − | <td>25 µL </td>
| |
| − | <td>0 µL </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>XbaI </sub></p></strong></td>
| |
| − | <td>1 µL </td>
| |
| − | <td>18 µL </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>SpeI</sub></p></strong></td>
| |
| − | <td>0.5 µL </td>
| |
| − | <td>9 µL </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>buffer 2.1</sub></p></strong></td>
| |
| − | <td>5 µL </td>
| |
| − | <td>90 µL </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td>
| |
| − | <td>18.5 µL </td>
| |
| − | <td>333 µL </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>Vol<sub>total</sub></p></strong></td>
| |
| − | <td>50 µL </td>
| |
| − | <td>450 µL </td>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | <center>Volumes</center></br></br></br>
| |
| − |
| |
| − |
| |
| − | 2. Incubate 10 min at 65°C to inactivate the enzymes.</br>
| |
| − | 3. Add 2 µL of rSAP in order to perform the dephosphorylation.</br>
| |
| − | 3. Store at -20°C</br></br>
| |
| − |
| |
| − | </p>
| |
| − | </figcaption>
| |
| − | </figure>
| |
| − | </div>
| |
| − |
| |
| − | <div class="lightbox" id="exp8">
| |
| − | <figure>
| |
| − | <a href="#" class="closemsg"></a>
| |
| − | <figcaption>
| |
| − | <p>
| |
| − | <U> Aim:</U> This step check the digestion efficiency of B1 (2 tubes)/B2 (2 tubes)/ C1 / C2 / A1 / A2 / D2 / E1 / E2.</br> Moreover, the inserts will be purified during this step because they will be extracted from the gel.</br>
| |
| − |
| |
| − | <U> Protocol:</U> follow in this link</br></br>
| |
| − | <U>What we did in the lab:</U></br>
| |
| − | <U>Materials:</U></br>
| |
| − | • Electrophoresis cuve</br>
| |
| − | • TAE 1X</br>
| |
| − | • Gene ruler (Thermoscientific 1kb plus)</br>
| |
| − | • Loading dye</br>
| |
| − | • Agarose</br>
| |
| − | • UV table </br>
| |
| − | • BET</br></br>
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| − |
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| − |
| |
| − | <U>Method:</U></br> Each well will contain 25 µL of DNA and 6 µL of Loading Dye. Follow the next deposit table :</br></br>
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| − |
| |
| − | Line 1:</br>
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| − | Ladder (6 µL) /// B2-col7 /// B2-col9 /// B1-col6 /// B1-col9 /// C1 /// C2</br></br>
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| − |
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| − | Line 2:</br>
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| − | Ladder (6 µL) /// D2 /// E1 /// E2 /// pSB1C€3 /// A1 /// A2</br></br>
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| − |
| |
| − | </p>
| |
| − | </figcaption>
| |
| − | </figure>
| |
| − | </div>
| |
| − |
| |
| − | <div class="lightbox" id="exp9">
| |
| − | <figure>
| |
| − | <a href="#" class="closemsg"></a>
| |
| − | <figcaption>
| |
| − | <p>
| |
| − | <U> Aim:</U> To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B1 (2 tubes)/B2 (2 tubes)/ C1 / C2 / A1 / A2 / D2 / E1 / E2 but we only extract B2 bands.</br> </br>
| |
| − | <U> Protocol:</U> follow in this link</br></br>
| |
| − | <U>What we did in the lab:</U></br>
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| − | <U>Materials:</U></br>
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| − | • scalpel</br>
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| − | • 2 ml eppendorfs</br>
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| − | • balance</br>
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| − | • UV table</br>
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| − | • microbiology equipment</br>
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| − | • QIAGEN Gel Extraction Kit</br>
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| − | </br></br>
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| − | <U>Method:</U></br>
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| − | Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection</br>
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| − |
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| − | Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer. </br></br>
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| − |
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| − | <table>
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| − | <thead>
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| − | <tr>
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| − | <th>Insert</th>
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| − | <th>Mass of gel (mg)</th>
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| − | <th>Volume of QG Buffer (µL )</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>B2-col7</p></strong></td>
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| − | <td>190</td>
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| − | <td>570</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B2-col9</p></strong></td>
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| − | <td>170</td>
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| − | <td>510</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B1-col6</p></strong></td>
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| − | <td>80</td>
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| − | <td>240</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B1-col8</p></strong></td>
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| − | <td>140</td>
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| − | <td>420</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>C1</p></strong></td>
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| − | <td>150</td>
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| − | <td>450</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>C2</p></strong></td>
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| − | <td>130</td>
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| − | <td>390</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>D2</p></strong></td>
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| − | <td>150</td>
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| − | <td>450</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>E1</p></strong></td>
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| − | <td>150</td>
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| − | <td>450</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>E2</p></strong></td>
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| − | <td>180</td>
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| − | <td>540</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>pSB1C3</p></strong></td>
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| − | <td>210</td>
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| − | <td>630</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A1</p></strong></td>
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| − | <td>170</td>
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| − | <td>510</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A2</p></strong></td>
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| − | <td>210</td>
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| − | <td>630</td>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | <center>Masses</center></br></br></br>
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| − |
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| − |
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| − |
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| − | <div class="lightbox" id="exp10">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
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| − | <figcaption>
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| − | <p>
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| − | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher)</br>
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| − |
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| − | <U>What we did in the lab:</U></br>
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| − | <U>Materials:</U></br>
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| − | • Nanodrop (Thermofisher)</br>
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| − | • Elution buffer from QIAGEN kit</br>
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| − | • Microbiology equipment (Follow this link)</br></br>
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| − |
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| − | <U>Method:</U></br>
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| − | Analyze absorbance at 260nm</br>
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| − | Clean the Nanodrop with water</br>
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| − | Make the blank with 1ul of elution buffer</br>
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| − | Put 1ul of your sample on the Nanodrop</br>
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| − | Make the measure and clean the Nanodrop between each measure</br></br>
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| − |
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| − | <U>Results:</U></br>
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| − |
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| − | <table>
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| − | <thead>
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| − | <tr>
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| − | <th>Absorbance at 260nm</th>
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| − | <th>A260/280</th>
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| − | <th>Concentration (ng/µL )</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <td><strong><p>B2-col7</p></strong></td>
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| − | <td>2.12</td>
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| − | <td>3.7</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B2-col9</p></strong></td>
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| − | <td>2.19</td>
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| − | <td>5.3</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B1-col6</p></strong></td>
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| − | <td>2.04</td>
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| − | <td>3.2</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>B1-col8</p></strong></td>
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| − | <td>1.98</td>
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| − | <td>3.7</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>C1</p></strong></td>
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| − | <td>1.78 </td>
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| − | <td>5.0</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>C2</p></strong></td>
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| − | <td>1.90 </td>
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| − | <td>307.1</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>D2</p></strong></td>
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| − | <td>1.84 </td>
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| − | <td>5.2</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>E1</p></strong></td>
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| − | <td>2.30 </td>
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| − | <td>3.9</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>E2</p></strong></td>
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| − | <td>2.17 </td>
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| − | <td>3.8</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>pSB1C3</p></strong></td>
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| − | <td>1.98</td>
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| − | <td>13.1</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A1</p></strong></td>
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| − | <td>2.11</td>
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| − | <td>4.8</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A2</p></strong></td>
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| − | <td>2.45</td>
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| − | <td>4.3</td>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | <center>Absorbance</center></br></br></br>
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| − |
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| − | </p>
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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| − |
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| − |
| |
| − | </body>
| |
| − | </html>
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