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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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| − | <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="week14">
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| − | <p><h5><B>September 6, 2016:</B></h5></p>
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| − | <p>
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| − | <a href="#exp1"> <h4>Growth of bacteria </h4></a></br>
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| − | </p>
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| − | <p><h5><B>September 7, 2016:</B></h5></p>
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| − | <p>
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| − | <a href="#exp2"><h4> Purification of the protein </h4></a></br>
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| − | </p>
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| − | <p><h5><B>September 8, 2016:</B></h5></p>
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| − | <p>
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| − | <a href="#exp3"><h4> Protein gel on SDS-Page </h4></a></br>
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| − | <a href="#exp4"><h4> Harvest the culture with Miniprep </h4></a></br>
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| − |
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| − | </p>
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| − | <p><h5><B>September 9, 2016:</B></h5></p>
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| − | <p>
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| − | <a href="#exp5"><h4> Extraction of plasmid DNA </h4></a></br>
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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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| − | </p>
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| − |
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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> Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an optical density of 0.7.</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>
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| − | • 2 2L erlenmeyers</br>
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| − | • LB (lysogeny Luria broth)</br>
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| − | • IPTG (0.1M)</br>
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| − | • Precultures in 25ml erlenmeyers</br>
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| − | • UV spectrophotometer (Ultrospec 3100)</br>
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| − | • Shaking incubator (INFORS HT)</br>
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| − | • Centrifuge</br>
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| − | • Buffer A (50mM Tris, 150mM of NaCl)</br> </br>
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| − |
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| − | <U>Method:</U></br>
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| − | Put 1L of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150RPM</br>
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| − | Once warmed, add 12.5ml of preculture in each erlenmeyer</br>
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| − | Let grow in the shaking incubator.</br>
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| − | Measure the absorbance with the UV spectrophotometer every 30min</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>Time</th>
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| − | <th>C2(1)</th>
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| − | <th>C2(2)</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>10:30AM</p></strong></td>
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| − | <td>0</td>
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| − | <td>0 </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>01:55PM</p></strong></td>
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| − | <td>0.438</td>
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| − | <td>/</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>2:25PM</p></strong></td>
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| − | <td>0.602</td>
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| − | <td>0.429 </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>2:45PM</p></strong></td>
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| − | <td>0.679</td>
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| − | <td>0.600 </td>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | <center>Optical Density</center></br></br></br>
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| − |
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| − |
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| − | 5. Add IPTG to reach a concentration of 0.1mM. (3:00PM)</br>
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| − | 6. The last measure before induction is pelleted 3min at 8000g and stored at -20°C.</br>
7. Let induce overnight in the shaking incubator at 15°C at 150RPM</br>
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| − | 8. The day after, measure the OD and store the measure pelleted at -20°C.</br>
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| − | 9. Centrifuge at 4500RPM the culture and throw out the supernatant, the pellet resuspended in 5ml of buffer A are stored at -80°C before protein extraction.</br> </br>
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| − |
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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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| − | </p>
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| − |
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| − |
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| − | <div class="lightbox" id="exp2">
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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> Purifying the protein C2 produced by BL21(DE3) using a Fast Purification Liquid Chromatography. We use the culture induced the previous day. </br> </br>
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| − |
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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>
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| − | • Fast Purification Liquid Chromatography </br>
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| − | • Chaotropic reagent (Guanidinium 6M) </br>
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| − | • EDTA 0,1M </br>
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| − | • PMSF (100mM) </br>
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| − | • Ni 2+ solution (100mM) </br>
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| − | • Centrifuge (labo deshmukh) </br>
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| − | • Buffer A (50mM Tris, 150mM of NaCl) </br>
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| − | • Buffer B (50mM Tris, 150mM of NaCl, 250mM Imidazole, pH=7.4) </br> </br>
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| − |
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| − | <U>Method:</U></br>
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| − | Melt the pellet of bacteria C2 (from 1L culture) and resuspend it with 10ml of buffer A. We had 25ml of pellet we complete until 40ml with buffer A. </br>
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| − | Add 40 µL of PMSF to avoid protein denaturation. </br>
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| − | Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A. </br>
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| − | Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication. </br>
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| − | Centrifuge 25 min at 16000g (rotor JA 25.50) </br>
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| − | Inject your sample in the FPLC </br>
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| − | Get back several samples: </br>
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| − | • C: Crude extract : before centrigugation </br>
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| − | • P: Pellet </br>
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| − | • SN: Supernatant </br>
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| − | • F: Flow through (unfixed proteins) </br>
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| − | • W: Wash 5% of buffer B </br>
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| − | • Fractions (depending on the gradient) </br> </br>
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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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| − |
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| − |
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| − |
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| − | <div class="lightbox" id="exp3">
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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> Get the size of the protein purified thanks to FPLC in order to know if it is our protein </br> </br>
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| − |
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| − | <U>Material: </U>
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| − | • SDS-Page cuve </br>
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| − | • SDS-Page gel (BIORAD) </br>
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| − | • Protein migration buffer </br>
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| − | • Protein ladder </br>
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| − | • Laemmli 2X </br>
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| − | • Coomassie Blue </br>
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| − | • Microbiology equipment (Follow this link) </br> </br>
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| − |
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| − | <U>Method:</U>
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| − | In 9 1.5ml eppendorf, put 20 µL of a sample and 20 µL of Laemmli 2X. </br>
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| − | Let denaturate the proteins 5min at 95°C </br>
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| − | Place the gel into the cuve and fill it with migration buffer </br>
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| − | Follow the next deposit table: </br>
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| − |
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| − | • Fraction 13 </br>
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| − | • Fraction 15 </br>
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| − | • Fraction 17 </br>
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| − | • Fraction 19 </br>
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| − | • Fraction 21 </br>
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| − | • Fraction 23 </br>
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| − | • Fraction 25 </br>
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| − | • Fraction 27 </br>
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| − | • Protein gene ruler (8 µL) </br>
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| − | 4. Launch the migration at 130V. </br>
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| − | 5. Wash the gel three times with distilled water during 5min. </br>
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| − | 6. Color the gel with Coomassie Blue diluted 1/5 during 30min. </br>
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| − | 7. Wash with distilled water for 5min then let wash 15min.
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| − | </br> </br>
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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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| − |
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| − |
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| − |
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| − | <div class="lightbox" id="exp4">
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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 start a culture for Miniprep of insert A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2 </br>
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| − | In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.</br> </br>
| |
| − | <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>
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| − | • Microbiology equipement </br>
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| − | • 15 mL Falcons </br>
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| − | • Carbenicillin 50 mg/ml </br>
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| − | • LB medium </br> </br>
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| − |
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| − | <U>Method:</U></br>
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| − | One colony is picked from the plates and shaken in 5.0 mL of LB supplemented with Carbenicillin at 50 µg/ml. </br>
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| − | The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br> </br>
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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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| − |
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| − | <div class="lightbox" id="exp5">
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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 perform a Midiprep to isolate plasmid DNA of pET43.1a with the inserts A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2 from cultures made on the 8/09. </br> The amplification method to increase the amount of plasmid is called Midiprep. </br>
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| − |
| |
| − | <U> Protocol:</U> follow in this link</br></br>
| |
| − | <U>What we did in the lab:</U></br>
| |
| − | <U>Materials:</U></br>
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| − | • 50 ml Falcon tube </br>
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| − | • Shaking incubator (INFORS HT) </br>
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| − | • Swing bucket centrifuge (JOUAN GR41) </br>
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| − | • QIAGEN Miniprep kit </br>
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| − | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) </br> </br>
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| − |
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| − | <U>Method:</U></br>
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| − | The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below. </br> </br>
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| − |
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| − |
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| − | Follow QIAGEN kit steps. Final volume: 100 µL </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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| − |
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| − | <div class="lightbox" id="exp6">
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| − | <figure>
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| − | <a href="#" class="closemsg"></a>
| |
| − | <figcaption>
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| − | <p>
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| − | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) </br> </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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| − |
| |
| − | <U>Results:</U></br>
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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</th>
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| − | <th>A280</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>A1</p></strong></td>
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| − | <td>0.645</td>
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| − | <td>0.345</td>
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| − | <td>1.87</td>
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| − | <td>32.3</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A1’ <sub>(diluted 1/2)</sub></p></strong></td>
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| − | <td>1.314</td>
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| − | <td>0.704</td>
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| − | <td>1.86 </td>
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| − | <td>65.7 </td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A2<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>1.404</td>
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| − | <td>0.752</td>
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| − | <td>1.87 </td>
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| − | <td>70.2</td>
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| − | </tr>
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| − | <tr>
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| − | <td><strong><p>A2’<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.494</td>
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| − | <td>0.269</td>
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| − | <td>1.87</td>
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| − | <td>24.7</td>
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| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>C1<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.305 </td>
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| − | <td>0.165</td>
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| − | <td>1.85 </td>
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| − | <td>15.2 </td>
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| − | </tr>
| |
| − | <tr>
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| − | <td><strong><p>C1’<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.812 </td>
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| − | <td>0.445</td>
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| − | <td>1.83</td>
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| − | <td>40.6</td>
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| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>C2<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.499 </td>
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| − | <td>0.272</td>
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| − | <td>1.83</td>
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| − | <td>24.9 </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>C2’<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.757 </td>
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| − | <td>0.402</td>
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| − | <td>1.88</td>
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| − | <td>37.8</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>D1<sub> (diluted 1/4)</sub></p></strong></td>
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| − | <td>0.441 </td>
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| − | <td>0.233</td>
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| − | <td>1.89</td>
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| − | <td>22.1 </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>D1’<sub> (diluted 1/4)</sub>)</p></strong></td>
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| − | <td>0.725</td>
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| − | <td>0.396</td>
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| − | <td>1.83</td>
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| − | <td>36.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>D2<sub> (diluted 1/4)</sub>)</p></strong></td>
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| − | <td>0.264 </td>
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| − | <td>0.141</td>
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| − | <td>1.87</td>
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| − | <td>13.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>D2’<sub> (diluted 1/4)</sub>)</p></strong></td>
| |
| − | <td>0.418 </td>
| |
| − | <td>0.216</td>
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| − | <td>1.94</td>
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| − | <td>20.9 </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>E1<sub> (diluted 1/4)</sub>)</p></strong></td>
| |
| − | <td>0.487 </td>
| |
| − | <td>0.266</td>
| |
| − | <td>1.83</td>
| |
| − | <td>24.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>E1’<sub> (diluted 1/4)</sub>)</p></strong></td>
| |
| − | <td>0.478 </td>
| |
| − | <td>0.247</td>
| |
| − | <td>1.94</td>
| |
| − | <td>24.0</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>E2<sub> (diluted 1/4)</sub>)</p></strong></td>
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| − | <td>1.364</td>
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| − | <td>0.742</td>
| |
| − | <td>1.84</td>
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| − | <td>68.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong><p>E2’<sub> (diluted 1/4)</sub>)</p></strong></td>
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| − | <td>1.041 </td>
| |
| − | <td>0.562</td>
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| − | <td>1.85</td>
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| − | <td>52.1</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | <center>Absorbance</center></br></br></br>
| |
| − |
| |
| − | </p>
| |
| − | </figcaption>
| |
| − | </figure>
| |
| − | </div>
| |
