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| | <div id="week9"> | | <div id="week9"> |
| | <p><h5><B>Week 9</B></h5></p> | | <p><h5><B>Week 9</B></h5></p> |
| − | <p><h3><B>August 29, 2016:</B></h3></p>
| + | |
| − | <p>
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| − | <a href="#exp1"><h4>Measure the amount of DNA extracted from the miniprep</h4></a></br>
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| − | </p>
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| − | <p><h3><B>September 1, 2016:</B></h3></p>
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| − | <p>
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| − | <a href="#exp2"><h4>Harvest the culture with Midiprep</h4></a></br>
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| − | </p>
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| − | <p><h3><B>September 2, 2016:</B></h3></p>
| + | |
| − | <p>
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| − | <a href="#exp3"><h4> Growth of bacteria </h4></a></br>
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| − | <a href="#exp4"><h4> Extraction of plasmid DNA </h4></a></br>
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| − | | + | |
| − | </p>
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| − | | + | |
| − | <div class="lightbox" id="exp1">
| + | |
| − | <figure>
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| − | <a href="#" class="closemsg"></a>
| + | |
| − | <figcaption>
| + | |
| − | <p>
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| − | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)</br></br>
| + | |
| − | <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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| − | • 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>
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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>
| + | |
| − | <tbody>
| + | |
| − | <tr>
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| − | <td><strong><p>A1(1)</p></strong></td>
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| − | <td>0.202 </td>
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| − | <td>0.124</td>
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| − | <td>1.62 </td>
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| − | <td>10.1</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>A1(2)</p></strong></td>
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| − | <td>0.259 </td>
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| − | <td>0.169</td>
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| − | <td>1.53 </td>
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| − | <td>13.0 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>A2(1)</p></strong></td>
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| − | <td>0.179</td>
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| − | <td>0.105</td>
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| − | <td>1.70 </td>
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| − | <td>8.9 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>A2(2)</p></strong></td>
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| − | <td>0.179 </td>
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| − | <td>0.103</td>
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| − | <td>1.73</td>
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| − | <td>8.9 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>A2(3)</p></strong></td>
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| − | <td>0.098 </td>
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| − | <td>0.052</td>
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| − | <td>1.86 </td>
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| − | <td>4.9 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>A2(4)</p></strong></td>
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| − | <td>0.152 </td>
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| − | <td>0.099</td>
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| − | <td>1.53</td>
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| − | <td>7.6 </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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| − | | + | |
| − | </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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| − | | + | |
| − | | + | |
| − | <div class="lightbox" id="exp2">
| + | |
| − | <figure>
| + | |
| − | <a href="#" class="closemsg"></a>
| + | |
| − | <figcaption>
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| − | <p><U> Aim:</U> To start a culture for Miniprep of insert C2 in pET43.1a in BL21(DE3) from 22/08 and A1/C2 in pET43.1a in TOP1O. </br>
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| − | In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.</br></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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| − | • Microbiology equipement </br>
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| − | • 50mL erlenmeyers</br>
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| − | • Carbenicillin 50 mg/ml</br>
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| − | • LB medium</br></br>
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| − | | + | |
| − | <U>Method:</U></br>
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| − | One colony is picked from the plates and shaken in 25.0 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>
| + | |
| − | </figcaption>
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| − | </figure>
| + | |
| − | </div>
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| − | | + | |
| − | <div class="lightbox" id="exp3">
| + | |
| − | <figure>
| + | |
| − | <a href="#" class="closemsg"></a>
| + | |
| − | <figcaption>
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| − | <p><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>
| + | |
| − | | + | |
| − | <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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| − | • 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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| − | | + | |
| − | <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>
| + | |
| − | | + | |
| − | <table>
| + | |
| − | <thead>
| + | |
| − | <tr>
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| − | <th>Time</th>
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| − | <th>C2(1)</th>
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| − | <th>C2(2)</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
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| − | <td><strong><p>9:36AM</p></strong></td>
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| − | <td>0.024 </td>
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| − | <td>0.023 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>10:06AM</p></strong></td>
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| − | <td>0.049</td>
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| − | <td>0.046 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>11:02AM</p></strong></td>
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| − | <td>0.175</td>
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| − | <td>0.165</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td><strong><p>11:36AM</p></strong></td>
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| − | <td>0.395 </td>
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| − | <td>0.402 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>12:05AM</p></strong></td>
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| − | <td>0.568</td>
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| − | <td>0.540 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>12:27AM</p></strong></td>
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| − | <td>0.658 </td>
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| − | <td>0.638</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>12:38AM</p></strong></td>
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| − | <td>0.694 </td>
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| − | <td>0.680 </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
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| − | <td><strong><p>14:31PM</p></strong></td>
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| − | <td>0.872 </td>
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| − | <td>0.859 </td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
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| − | <center>Optical Density</center></br></br></br>
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| − | 5. Add IPTG to reach a concentration of 0.1mM. (12:57AM)</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</br>
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| − | 8. After 7 hours of induction, 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.
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| − | </br></br>
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| − | </p>
| + | |
| − | </figcaption>
| + | |
| − | </figure>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="lightbox" id="exp4">
| + | |
| − | <figure>
| + | |
| − | <a href="#" class="closemsg"></a>
| + | |
| − | <figcaption>
| + | |
| − | <p>
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| − | <U> Aim:</U> To perform a Midiprep to isolate plasmid DNA of pET43.1a with the inserts A1/C2 from cultures made on the 1/09. </br>The amplification method to increase the amount of plasmid is called Midiprep.</br></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>
| + | |
| − | • 50 ml Falcon tube</br>
| + | |
| − | • Shaking incubator (INFORS HT)</br>
| + | |
| − | • Swing bucket centrifuge (JOUAN GR41)</br>
| + | |
| − | • QIAGEN Miniprep kit</br>
| + | |
| − | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)</br></br>
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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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| − | | + | |
| − | | + | |
| − | Follow QIAGEN kit steps. Final volume: 100 µL</br></br>
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| − | | + | |
| − | </p>
| + | |
| − | </figcaption>
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| − | </figure>
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| | </div> | | </div> |
