Difference between revisions of "Team:Pasteur Paris/Microbiology week13"
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<p><h3><B>August 29, 2016:</B></h3></p> | <p><h3><B>August 29, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp1"><h4>Measure the amount of DNA extracted from the miniprep</h4></a>< | + | <a href="#exp1"><h4> 232. Measure the amount of DNA extracted from the miniprep</h4></a><br/> |
</p> | </p> | ||
<p><h3><B>September 1, 2016:</B></h3></p> | <p><h3><B>September 1, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp2"><h4>Harvest the culture with Midiprep</h4></a>< | + | <a href="#exp2"><h4> 233. Harvest the culture with Midiprep</h4></a><br/> |
</p> | </p> | ||
<p><h3><B>September 2, 2016:</B></h3></p> | <p><h3><B>September 2, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp3"><h4> Growth of bacteria </h4></a>< | + | <a href="#exp3"><h4> 234. Growth of bacteria </h4></a><br/> |
| − | <a href="#exp4"><h4> Extraction of plasmid DNA </h4></a>< | + | <a href="#exp4"><h4> 235. Extraction of plasmid DNA </h4></a><br/> |
</p> | </p> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)< | + | <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)<br/><br/> |
| − | + | <U>What we did in the lab:</U><br/> | |
| − | <U>What we did in the lab:</U></ | + | <U>Materials:</U><br/> |
| − | <U>Materials:</U>< | + | • Nanodrop (Thermofisher)<br/> |
| − | • Nanodrop (Thermofisher)< | + | • Elution buffer from QIAGEN kit<br/> |
| − | • Elution buffer from QIAGEN kit< | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)<br/><br/> |
| − | • Microbiology equipment (Follow this link | + | |
| − | <U>Method:</U>< | + | <U>Method:</U><br/> |
| − | Analyze absorbance at 260nm< | + | 1. Analyze absorbance at 260nm<br/> |
| − | Clean the Nanodrop with water< | + | 2. Clean the Nanodrop with water<br/> |
| − | Make the blank with | + | 3. Make the blank with 1µl of elution buffer<br/> |
| − | Put | + | 4. Put 1 µl of your sample on the Nanodrop<br/> |
| − | Make the measure and clean the Nanodrop between each measure< | + | 5. Make the measure and clean the Nanodrop between each measure <br/><br/> |
| − | <U>Results:</U>< | + | <U>Results:</U><br/> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p>Table 1 : Absorbance</p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th>Absorbance at 260nm</th> | <th>Absorbance at 260nm</th> | ||
| − | <th> | + | <th>A<sub>260</sub></th> |
| − | <th> | + | <th>A<sub>280</sub></th> |
| − | <th> | + | <th>A<sub>260/280</sub<</th> |
| − | <th>Concentration (ng/µ | + | <th>Concentration (ng/µl)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A1(1)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A1(1)</p></strong></td> |
| − | <td>0.202 </td> | + | <td align="center"; valign="center">0.202 </td> |
| − | <td>0.124</td> | + | <td align="center"; valign="center">0.124</td> |
| − | <td>1.62 </td> | + | <td align="center"; valign="center">1.62 </td> |
| − | <td>10.1</td> | + | <td align="center"; valign="center">10.1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A1(2)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A1(2)</p></strong></td> |
| − | <td>0.259 </td> | + | <td align="center"; valign="center">0.259 </td> |
| − | <td>0.169</td> | + | <td align="center"; valign="center">0.169</td> |
| − | <td>1.53 </td> | + | <td align="center"; valign="center">1.53 </td> |
| − | <td>13.0 </td> | + | <td align="center"; valign="center">13.0 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2(1)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2(1)</p></strong></td> |
| − | <td>0.179</td> | + | <td align="center"; valign="center">0.179</td> |
| − | <td>0.105</td> | + | <td align="center"; valign="center">0.105</td> |
| − | <td>1.70 </td> | + | <td align="center"; valign="center">1.70 </td> |
| − | <td>8.9 </td> | + | <td align="center"; valign="center">8.9 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2(2)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2(2)</p></strong></td> |
| − | <td>0.179 </td> | + | <td align="center"; valign="center">0.179 </td> |
| − | <td>0.103</td> | + | <td align="center"; valign="center">0.103</td> |
| − | <td>1.73</td> | + | <td align="center"; valign="center">1.73</td> |
| − | <td>8.9 </td> | + | <td align="center"; valign="center">8.9 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2(3)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2(3)</p></strong></td> |
| − | <td>0.098 </td> | + | <td align="center"; valign="center">0.098 </td> |
| − | <td>0.052</td> | + | <td align="center"; valign="center">0.052</td> |
| − | <td>1.86 </td> | + | <td align="center"; valign="center">1.86 </td> |
| − | <td>4.9 </td> | + | <td align="center"; valign="center">4.9 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2(4)</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2(4)</p></strong></td> |
| − | <td>0.152 </td> | + | <td align="center"; valign="center">0.152 </td> |
| − | <td>0.099</td> | + | <td align="center"; valign="center">0.099</td> |
| − | <td>1.53</td> | + | <td align="center"; valign="center">1.53</td> |
| − | <td>7.6 </td> | + | <td align="center"; valign="center">7.6 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br/> |
| − | < | + | <br/><br/><br/> |
</p> | </p> | ||
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<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <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. < | + | <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/> |
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. | In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. | ||
| − | </br></br> | + | <br/><br/> |
| + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br/><br/> | ||
| + | <U>What we did in the lab:</U><br/> | ||
| + | <U>Materials:</U><br/> | ||
| + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br/> | ||
| + | • 50 ml erlenmeyers<br/> | ||
| + | • Carbenicillin 50 mg/ml<br/> | ||
| + | • LB medium<br/><br/> | ||
| − | <U> | + | <U>Method:</U><br/> |
| − | + | 1. One colony is picked from the plates and shaken in 25.0 ml of LB supplemented with Carbenicillin at 50 µg/ml.<br/> | |
| − | + | 2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.<br/> | |
| − | + | <br/><br/><br/> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | One colony is picked from the plates and shaken in 25.0 | + | |
| − | The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. | + | |
| − | < | + | |
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
| Line 359: | Line 362: | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <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> | + | <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 <a href="https://static.igem.org/mediawiki/2016/a/a4/T--Pasteur_Paris--Protein_induction_protocol.pdf">link</a><br/><br/> | ||
| + | <U>What we did in the lab:</U><br/> | ||
| + | <U>Materials:</U><br/> | ||
| + | • Two 2 l erlenmeyers<br/> | ||
| + | • LB (Luria broth)<br/> | ||
| + | • IPTG (0.1 M)<br/> | ||
| + | • Precultures in 25 ml Erlenmeyers<br/> | ||
| + | • UV spectrophotometer (Ultrospec 3100)<br/> | ||
| + | • Shaking incubator (INFORS HT)<br/> | ||
| + | • Centrifuge<br/> | ||
| + | • Buffer A (50mM Tris, 150mM of NaCl)<br/><br/> | ||
| − | <U> | + | <U>Method:</U><br/> |
| − | + | 1. Put 1 l of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150 rpm.<br/> | |
| − | + | 2. Once warmed, add 12.5 ml of preculture in each erlenmeyer.<br/> | |
| − | + | 3. Let grow in the shaking incubator.<br/> | |
| − | + | 4. Measure the absorbance with the UV spectrophotometer every 30min<br/> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Once warmed, add 12. | + | |
| − | Let grow in the shaking incubator.< | + | |
| − | Measure the absorbance with the UV spectrophotometer every 30min< | + | |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 140 :</U> Optical Density</p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th>Time</th> | <th>Time</th> | ||
| − | <th>C2(1)</th> | + | <th>C2 (1)</th> |
| − | <th>C2(2)</th> | + | <th>C2 (2)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>9: | + | <td align="center"; valign="center"><strong><p>9:36<sub>AM</sub></p></strong></td> |
| − | <td>0.024 </td> | + | <td align="center"; valign="center">0.024 </td> |
| − | <td>0.023 </td> | + | <td align="center"; valign="center">0.023 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>10: | + | <td align="center"; valign="center"><strong><p>10:06<sub>AM</sub></p></strong></td> |
| − | <td>0.049</td> | + | <td align="center"; valign="center">0.049</td> |
| − | <td>0.046 </td> | + | <td align="center"; valign="center">0.046 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>11: | + | <td align="center"; valign="center"><strong><p>11:02<sub>AM</sub></p></strong></td> |
| − | <td>0.175</td> | + | <td align="center"; valign="center">0.175</td> |
| − | <td>0.165</td> | + | <td align="center"; valign="center">0.165</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>11: | + | <td align="center"; valign="center"><strong><p>11:36<sub>AM</sub></p></strong></td> |
| − | <td>0.395 </td> | + | <td align="center"; valign="center">0.395 </td> |
| − | <td>0.402 </td> | + | <td align="center"; valign="center">0.402 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>12: | + | <td align="center"; valign="center"><strong><p>12:05<sub>AM</sub></p></strong></td> |
| − | <td>0.568</td> | + | <td align="center"; valign="center">0.568</td> |
| − | <td>0.540 </td> | + | <td align="center"; valign="center">0.540 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>12: | + | <td align="center"; valign="center"><strong><p>12:27<sub>AM</sub></p></strong></td> |
| − | <td>0.658 </td> | + | <td align="center"; valign="center">0.658 </td> |
| − | <td>0.638</td> | + | <td align="center"; valign="center">0.638</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>12: | + | <td align="center"; valign="center"><strong><p>12:38<sub>AM</sub></p></strong></td> |
| − | <td>0.694 </td> | + | <td align="center"; valign="center">0.694 </td> |
| − | <td>0.680 </td> | + | <td align="center"; valign="center">0.680 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>14: | + | <td align="center"; valign="center"><strong><p>14:31<sub>PM</sub></p></strong></td> |
| − | <td>0.872 </td> | + | <td align="center"; valign="center">0.872 </td> |
| − | <td>0.859 </td> | + | <td align="center"; valign="center">0.859 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br> |
| − | + | ||
| − | + | 5. Add IPTG to reach a concentration of 0.1mM. (12:57<sub>AM</sub>)<br/> | |
| − | 5. Add IPTG to reach a concentration of 0.1mM. (12: | + | 6. The last measure before induction is pelleted 3min at 8000g and stored at -20°C.<br/>
|
| − | 6. The last measure before induction is pelleted 3min at 8000g and stored at -20°C.< | + | 7. Let induce overnight in the shaking incubator<br/> |
| − | 8. After 7 hours of induction, measure the OD and store the measure pelleted at -20°C.< | + | 8. After 7 hours of induction, measure the OD<sub>600 nm</sub> and store the measure pelleted at -20°C.<br/> |
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. | 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/><br/> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
| + | |||
<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
| Line 448: | Line 453: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> To perform a Midiprep to isolate plasmid DNA of pET43. | + | <U> Aim:</U> To perform a Midiprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1/C2 from cultures made on the 1<sup>set</sup> of August. <br/> |
| − | + | The amplification method to increase the amount of plasmid is called Midiprep.<br/><br/> | |
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br/><br/> | |
| − | <U>What we did in the lab:</U>< | + | <U>What we did in the lab:</U><br/> |
| − | <U>Materials:</U>< | + | <U>Materials:</U><br/> |
| − | • 50 ml Falcon tube</ | + | • 50 ml Falcon tube<br/> |
| − | • Shaking incubator (INFORS HT)< | + | • Shaking incubator (INFORS HT)<br/> |
| − | • Swing bucket centrifuge (JOUAN GR41)< | + | • Swing bucket centrifuge (JOUAN GR41)<br/> |
• QIAGEN Miniprep kit</br> | • QIAGEN Miniprep kit</br> | ||
| − | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)<br/><br/> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | <U>Method:</U><br/> | ||
| + | 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/> | ||
| + | Follow QIAGEN kit steps with a final volume of 100 µl<br/> | ||
| + | <br/><br/><br/> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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</div> | </div> | ||
| − | + | ||
| − | + | ||
</body> | </body> | ||
</html> | </html> | ||
