Difference between revisions of "Team:Pasteur Paris/Microbiology week15"
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<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| − | • Restriction enzymes: | + | • Restriction enzymes: Xba I, Spe I (New England Biolabs, NEB)</br> |
• Restriction enzyme buffers </br> | • Restriction enzyme buffers </br> | ||
• 37°C water bath</br> | • 37°C water bath</br> | ||
| Line 269: | Line 270: | ||
<table> | <table> | ||
| − | + | <caption align="bottom" align="center"><i><p> <U>Table 143</U></p></i></caption> | |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 324: | Line 325: | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Analyze absorbance at | + | 1. Analyze absorbance at 260 nm</br> |
2. Clean the Nanodrop with water</br> | 2. Clean the Nanodrop with water</br> | ||
| − | 3. Make the blank with | + | 3. Make the blank with 1μl of elution buffer</br> |
| − | 4. Put | + | 4. Put 1μl of your sample on the Nanodrop</br> |
5. Make the measure and clean the Nanodrop between each measure</br></br> | 5. Make the measure and clean the Nanodrop between each measure</br></br> | ||
<U>Results:</U></br> | <U>Results:</U></br> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center"><i><p> Table | + | <caption align="bottom" align="center"><i><p> <U>Table 144 :</U> Absorbance </p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 390: | Line 391: | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| − | • Restriction enzymes: | + | • Restriction enzymes: Xba I, Spe I (New England Biolabs, NEB)</br> |
• Restriction enzyme buffers </br> | • Restriction enzyme buffers </br> | ||
• 37°C water bath</br> | • 37°C water bath</br> | ||
| Line 402: | Line 403: | ||
<table> | <table> | ||
| − | + | <caption align="bottom" align="center"><i><p> <U>Table 145</U></p></i></caption> | |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 417: | Line 418: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol<sub>XbaI </sub></p></strong></td> | + | <td align="center"; valign="center"><strong><p>Vol<sub>XbaI </sub></p></strong></td> |
| − | <td>1 µl </td> | + | <td align="center"; valign="center">1 µl </td> |
| − | <td>18 µl </td> | + | <td align="center"; valign="center">18 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol<sub>SpeI</sub></p></strong></td> | + | <td align="center"; valign="center"><strong><p>Vol<sub>SpeI</sub></p></strong></td> |
| − | <td>0.5 µ | + | <td align="center"; valign="center">0.5 µl </td> |
| − | <td>9 µ | + | <td align="center"; valign="center">9 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol<sub>buffer 2.1</sub></p></strong></td> | + | <td align="center"; valign="center"><strong><p>Vol<sub>buffer 2.1</sub></p></strong></td> |
| − | <td>5 µ | + | <td align="center"; valign="center">5 µl </td> |
| − | <td>90 µ | + | <td align="center"; valign="center">90 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td> | + | <td align="center"; valign="center"><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td> |
| − | <td>18.5 µ | + | <td align="center"; valign="center">18.5 µl </td> |
| − | <td>333 µ | + | <td align="center"; valign="center">333 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol<sub>total</sub></p></strong></td> | + | <td align="center"; valign="center"><strong><p>Vol<sub>total</sub></p></strong></td> |
| − | <td>50 µ | + | <td align="center"; valign="center">50 µl </td> |
| − | <td>450 µ | + | <td align="center"; valign="center">450 µl </td> |
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 2. Incubate 10 min at | + | 2. Incubate 10 min at 65°C to inactivate the enzymes.</br> |
| − | 3. Add 2 µ | + | 3. Add 2 µl of rSAP in order to perform the dephosphorylation.</br> |
| − | 3. Store at - | + | 3. Store at -20°C</br> |
| − | + | <br/><br/><br/> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
| Line 457: | Line 460: | ||
<figcaption> | <figcaption> | ||
<p> | <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> 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><br/> |
<U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| Line 467: | Line 470: | ||
• Agarose</br> | • Agarose</br> | ||
• UV table </br> | • UV table </br> | ||
| − | • | + | • Ethidium bromide drops (EB)</br></br> |
| − | <U>Method:</U></br> Each well will contain 25 µ | + | <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> |
Line 1:</br> | Line 1:</br> | ||
| − | Ladder (6 µ | + | Ladder (6 µl) | Ø | B2-col7 | Ø | B2-col9 | Ø | B1-col6 | Ø | B1-col9 | Ø | C1 | Ø | C2</br></br> |
Line 2:</br> | Line 2:</br> | ||
| − | Ladder (6 µ | + | Ladder (6 µl) | Ø | D2 | Ø | E1 | Ø | E2 | Ø | pSB1C€3 | Ø | A1 | Ø | A2</br></br> |
</p> | </p> | ||
| Line 482: | Line 485: | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
| + | |||
<div class="lightbox" id="exp5"> | <div class="lightbox" id="exp5"> | ||
| Line 492: | Line 498: | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| − | • | + | • Scalpel</br> |
| − | • 2 ml | + | • 2 ml Eppendorfs</br> |
| − | • | + | • Balance</br> |
• UV table</br> | • UV table</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>) |
• QIAGEN Gel Extraction Kit</br> | • QIAGEN Gel Extraction Kit</br> | ||
</br></br> | </br></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection</br> | + |   ⚠ Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection</br> |
Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer. </br></br> | Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer. </br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 146 :</U> Masses </p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Mass of gel (mg)</th> | <th>Mass of gel (mg)</th> | ||
| − | <th>Volume of QG Buffer (µ | + | <th>Volume of QG Buffer (µl)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B2-col7</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B2-col7</p></strong></td> |
| − | <td>190</td> | + | <td align="center"; valign="center">190</td> |
| − | <td>570</td> | + | <td align="center"; valign="center">570</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B2-col9</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B2-col9</p></strong></td> |
| − | <td>170</td> | + | <td align="center"; valign="center">170</td> |
| − | <td>510</td> | + | <td align="center"; valign="center">510</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B1-col6</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B1-col6</p></strong></td> |
| − | <td>80</td> | + | <td align="center"; valign="center">80</td> |
| − | <td>240</td> | + | <td align="center"; valign="center">240</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B1-col8</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B1-col8</p></strong></td> |
| − | <td>140</td> | + | <td align="center"; valign="center">140</td> |
| − | <td>420</td> | + | <td align="center"; valign="center">420</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>C1</p></strong></td> |
| − | <td>150</td> | + | <td align="center"; valign="center">150</td> |
| − | <td>450</td> | + | <td align="center"; valign="center">450</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>C2</p></strong></td> |
| − | <td>130</td> | + | <td align="center"; valign="center">130</td> |
| − | <td>390</td> | + | <td align="center"; valign="center">390</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>D2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>D2</p></strong></td> |
| − | <td>150</td> | + | <td align="center"; valign="center">150</td> |
| − | <td>450</td> | + | <td align="center"; valign="center">450</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>E1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>E1</p></strong></td> |
| − | <td>150</td> | + | <td align="center"; valign="center">150</td> |
| − | <td>450</td> | + | <td align="center"; valign="center">450</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>E2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>E2</p></strong></td> |
| − | <td>180</td> | + | <td align="center"; valign="center">180</td> |
| − | <td>540</td> | + | <td align="center"; valign="center">540</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pSB1C3</p></strong></td> | + | <td align="center"; valign="center"><strong><p>pSB1C3</p></strong></td> |
| − | <td>210</td> | + | <td align="center"; valign="center">210</td> |
| − | <td>630</td> | + | <td align="center"; valign="center">630</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A1</p></strong></td> |
| − | <td>170</td> | + | <td align="center"; valign="center">170</td> |
| − | <td>510</td> | + | <td align="center"; valign="center">510</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2</p></strong></td> |
| − | <td>210</td> | + | <td align="center"; valign="center">210</td> |
| − | <td>630</td> | + | <td align="center"; valign="center">630</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | <br/><br/><br/> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 593: | Line 600: | ||
• Nanodrop (Thermofisher)</br> | • Nanodrop (Thermofisher)</br> | ||
• Elution buffer from QIAGEN kit</br> | • Elution buffer from QIAGEN kit</br> | ||
| − | • Microbiology equipment (Follow this link | + | • 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> | <U>Method:</U></br> | ||
| − | Analyze absorbance at 260nm</br> | + | 1. Analyze absorbance at 260nm</br> |
| − | Clean the Nanodrop with water</br> | + | 2. Clean the Nanodrop with water</br> |
| − | Make the blank with | + | 3. Make the blank with 1μl of elution buffer</br> |
| − | Put 1ul of your sample on the Nanodrop</br> | + | 4. Put 1ul of your sample on the Nanodrop</br> |
| − | Make the measure and clean the Nanodrop between each measure</br></br> | + | 5. Make the measure and clean the Nanodrop between each measure</br></br> |
<U>Results:</U></br> | <U>Results:</U></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 147 :</U> Absorbance </p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| − | <th>Absorbance at | + | <th>Absorbance at 260 nm</th> |
<th>A260/280</th> | <th>A260/280</th> | ||
| − | <th>Concentration (ng/µ | + | <th>Concentration (ng/µl)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B2-col7</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B2-col7</p></strong></td> |
| − | <td>2.12</td> | + | <td align="center"; valign="center">2.12</td> |
| − | <td>3.7</td> | + | <td align="center"; valign="center">3.7</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B2-col9</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B2-col9</p></strong></td> |
| − | <td>2.19</td> | + | <td align="center"; valign="center">2.19</td> |
<td>5.3</td> | <td>5.3</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B1-col6</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B1-col6</p></strong></td> |
| − | <td>2.04</td> | + | <td align="center"; valign="center">2.04</td> |
| − | <td>3.2</td> | + | <td align="center"; valign="center">3.2</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>B1-col8</p></strong></td> | + | <td align="center"; valign="center"><strong><p>B1-col8</p></strong></td> |
| − | <td>1.98</td> | + | <td align="center"; valign="center">1.98</td> |
| − | <td>3.7</td> | + | <td align="center"; valign="center">3.7</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>C1</p></strong></td> |
| − | <td>1.78 </td> | + | <td align="center"; valign="center">1.78 </td> |
| − | <td>5.0</td> | + | <td align="center"; valign="center">5.0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>C2</p></strong></td> |
| − | <td>1.90 </td> | + | <td align="center"; valign="center">1.90 </td> |
| − | <td>307.1</td> | + | <td align="center"; valign="center">307.1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>D2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>D2</p></strong></td> |
| − | <td>1.84 </td> | + | <td align="center"; valign="center">1.84 </td> |
| − | <td>5.2</td> | + | <td align="center"; valign="center">5.2</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>E1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>E1</p></strong></td> |
| − | <td>2.30 </td> | + | <td align="center"; valign="center">2.30 </td> |
| − | <td>3.9</td> | + | <td align="center"; valign="center">3.9</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>E2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>E2</p></strong></td> |
| − | <td>2.17 </td> | + | <td align="center"; valign="center">2.17 </td> |
| − | <td>3.8</td> | + | <td align="center"; valign="center">3.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pSB1C3</p></strong></td> | + | <td align="center"; valign="center"><strong><p>pSB1C3</p></strong></td> |
| − | <td>1.98</td> | + | <td align="center"; valign="center">1.98</td> |
| − | <td>13.1</td> | + | <td align="center"; valign="center">13.1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A1</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A1</p></strong></td> |
| − | <td>2.11</td> | + | <td align="center"; valign="center">2.11</td> |
| − | <td>4.8</td> | + | <td align="center"; valign="center">4.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A2</p></strong></td> | + | <td align="center"; valign="center"><strong><p>A2</p></strong></td> |
| − | <td>2.45</td> | + | <td align="center"; valign="center">2.45</td> |
| − | <td>4.3</td> | + | <td align="center"; valign="center">4.3</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br/> |
| − | + | </br></br></br> | |
</p> | </p> | ||
