Difference between revisions of "Team:Pasteur Paris/Microbiology week3"
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| − | <div> | + | <div> |
| − | <h2><B>Microbiology Notebook</B></h2> | + | <h2><B>Microbiology Notebook</B></h2> |
| − | + | <div id="week3"> | |
| − | <p><h5><B>Week 3</B></h5></p> | + | <p><h5><B>Week 3</B></h5></p> |
| − | + | <p><h3><B> June 20, 2016:</B></h3></p> | |
| − | + | <p> | |
| − | + | <a href="#exp1"><h4> Dosage of pET43.1</h4></a></br> | |
| − | + | <a href="#exp2"><h4> Checkin of stab cultures</h4></a></br> | |
| − | + | <a href="#exp3"><h4> Transformation of pET43.1a (+)</h4></a></br> | |
| − | </p> | + | </p> |
| − | + | <p><h3><B> June 21, 2016:</B></h3></p> | |
| − | + | <p> | |
| − | + | <a href="#exp4"><h4> Analyse </h4></a></br> | |
| − | + | <a href="#exp5"><h4> Dosage of pET43.1a(+) </h4></a></br> | |
| − | + | <a href="#exp6"><h4> Digestion of pET43.1 and C1 and C2</h4></a></br> | |
| − | + | <a href="#exp7"><h4> Electrophoresis of pET43.1a(+) and the inserts C1 and C2:</h4></a></br> | |
| − | + | <a href="#exp8"><h4> Gel extraction:</h4></a></br> | |
| − | + | <a href="#exp9"><h4> Dephosphorylation of pET43.1a(+):</h4></a></br> | |
| − | + | </p> | |
| − | + | <p><h3><B> June 22, 2016:</B></h3></p> | |
| − | + | <p> | |
| − | + | <a href="#exp10"><h4> Ligation of pET43.1a(+) with C1 and C2 </h4></a></br> | |
| − | + | <a href="#exp11"><h4> Transformation of pET43.1a(+) linked with C1 and C2</h4></a></br> | |
| − | + | </p> | |
| − | + | <p><B><h3> June 23, 2016:</B></h3></p> | |
| − | + | <p> | |
| − | + | <a href="#exp12"><h4 Dosage of the uncut inserts</h4></a></br> | |
| − | + | </p> | |
| − | + | <div class="lightbox" id="exp12"> | |
| − | + | <figure> | |
| − | + | <a href="#" class="closemsg"></a> | |
| − | + | <figcaption> | |
<p> | <p> | ||
| − | + | <U> Aim:</U> On the June 6, 2016, we have done a transformation of pET43.1a(+) into DH5alpha and we dose it to know it concentration. </br></br> | |
| − | + | <U>What we did in the lab:</U></br> | |
| − | + | <U>Materials:</U></br> | |
| − | <U>Method:</U></br> The culture for plasmid amplification was carried out in a large volume then aliquoted in several 1.5 ml Eppendorfs for centrifugation, since our Falcon size rotors do not go up high in G force. </br></br> | + | <U>Method:</U></br> The culture for plasmid amplification was carried out in a large volume then aliquoted in several 1.5 ml Eppendorfs for centrifugation, since our Falcon size rotors do not go up high in G force. </br></br> |
We have nine 1 ml Eppendorf with 50L of pET43.1 in each tubes so we assemble all tubes in one. </br> | We have nine 1 ml Eppendorf with 50L of pET43.1 in each tubes so we assemble all tubes in one. </br> | ||
1. Centrifuge the first Eppendorf and pour it into the second Eppendorf. </br> | 1. Centrifuge the first Eppendorf and pour it into the second Eppendorf. </br> | ||
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4. Put in 1ml of TE1X with P1000, then take off 15L with P20 and add 15L of DNA with P20. </br> | 4. Put in 1ml of TE1X with P1000, then take off 15L with P20 and add 15L of DNA with P20. </br> | ||
5. Analysis in spectrophotometer, Eppendorf biophotometer plus at 260 nm</br></br> | 5. Analysis in spectrophotometer, Eppendorf biophotometer plus at 260 nm</br></br> | ||
| − | + | <U> Results: </U></br> | |
| − | + | C1=87 ng/L</br> | |
| − | <U> Results: </U></br> | + | C2 = 103.3 ng/L</br></br> |
| − | C1=87 ng/L</br> | + | |
| − | C2 = 103.3 ng/L</br></br> | + | |
Due to these bad results, we decided to use another spectrophotometer “Eppendorf Biophotometer”:</br> | Due to these bad results, we decided to use another spectrophotometer “Eppendorf Biophotometer”:</br> | ||
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Again, our results were not correct so we remeasure with utraspec 3100 pro from Amersham Bioscience. </br> | Again, our results were not correct so we remeasure with utraspec 3100 pro from Amersham Bioscience. </br> | ||
| − | + | <table> | |
| − | + | <thead> | |
| − | + | <tr> | |
| − | + | <th></th> | |
| − | + | <th>Sample 1</th> | |
| − | + | <th>Sample 2</th> | |
| − | + | <th>Sample 3</th> | |
| − | + | </tr> | |
| − | + | </thead> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <tbody> | |
| − | + | <tr> | |
| − | + | <td><strong><p>A<sub>260</sub></p></strong></td> | |
| − | + | <td>0.043 </td> | |
| − | + | <td>0.59 </td> | |
| − | + | <td>0.037 </td> | |
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>A<sub>280</sub></p></strong></td> | ||
| + | <td>0.026 </td> | ||
| + | <td>0.01 </td> | ||
| + | <td>0.016 </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> | ||
| + | <td>1.7 </td> | ||
| + | <td>5.9 </td> | ||
| + | <td>2.31 </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> | ||
| + | <td>2.15 </td> | ||
| + | <td>1.95 </td> | ||
| + | <td>1.85 </td> | ||
| + | |||
| + | </tr> | ||
| − | + | <tr> | |
| − | + | <td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | |
| − | + | <td>143.33 </td> | |
| − | + | <td>130 </td> | |
| − | + | <td>123.33 </td> | |
| − | + | ||
| − | </tr> | + | </tr> |
| − | + | ||
| − | + | <tr> | |
| − | + | <td><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> | |
| + | <td>133.22 </td> | ||
| + | <td>133.22 </td> | ||
| + | <td>133.22 </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center> Table 10</center></br></br></br> | ||
We keep these results for the end of the experiment. </br> | We keep these results for the end of the experiment. </br> | ||
| − | Then, we resuspend the insert by adding buffer TE1X in each tube (refer to the June 15, 2016 for the volumes). | + | Then, we resuspend the insert by adding buffer TE1X in each tube (refer to the June 15, 2016 for the volumes).</br></br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp2"> | <div class="lightbox" id="exp2"> | ||
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</div> | </div> | ||
| − | div class="lightbox" id="exp4"> | + | <div class="lightbox" id="exp4"> |
<figure> | <figure> | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Results:</U> We analyse the transformation done yesterday. We don’t saw bacteria colonies on the dish, so transformations were not successful. To understand why bacterial colonies don’t grow up on petri dish, plasmid DNA is assayed. | + | <p><U> Results:</U> We analyse the transformation done yesterday. We don’t saw bacteria colonies on the dish, so transformations were not successful. To understand why bacterial colonies don’t grow up on petri dish, plasmid DNA is assayed.</br> </br> |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
| − | + | </div> | |
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2. We made a 15/1000 dilutions with our samples (15 L of DNA and 985L of water) </br> | 2. We made a 15/1000 dilutions with our samples (15 L of DNA and 985L of water) </br> | ||
3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back 15L and add of DNA</br> | 3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back 15L and add of DNA</br> | ||
| − | 4. We analyse at 260 nm</br> | + | 4. We analyse at 260 nm</br></br></br> |
| − | </br></br> | + | |
<table> | <table> | ||
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</tr> | </tr> | ||
</thead> | </thead> | ||
| + | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
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<td></td> | <td></td> | ||
</tr> | </tr> | ||
| + | |||
<tr> | <tr> | ||
<td><strong><p> A<sub>280</sub> </p></strong></td> | <td><strong><p> A<sub>280</sub> </p></strong></td> | ||
| − | + | <td></td> | |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| + | |||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260</span> | + | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> |
<td></td> | <td></td> | ||
<td> </td> | <td> </td> | ||
<td> </td> | <td> </td> | ||
</tr> | </tr> | ||
| + | |||
<tr> | <tr> | ||
<td><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> | <td><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> | ||
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</tr> | </tr> | ||
| + | |||
<tr> | <tr> | ||
<td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | <td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | ||
<td>250 </td> | <td>250 </td> | ||
<td>200 </td> | <td>200 </td> | ||
| − | </tr> | + | </tr> |
| + | |||
<tr> | <tr> | ||
<td><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> | <td><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> | ||
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<td>225 </td> | <td>225 </td> | ||
<td>225 </td> | <td>225 </td> | ||
| − | </tr> | + | </tr> |
</tbody> | </tbody> | ||
</table> | </table> | ||
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<p> | <p> | ||
<U> Aim:</U> Our transformation do not work so we made a new digestion with BamHI and Hind III. This step will let us to do the transformation..</br> | <U> Aim:</U> Our transformation do not work so we made a new digestion with BamHI and Hind III. This step will let us to do the transformation..</br> | ||
| − | + | <U> Protocol:</U> follow in this link</br></br> | |
| − | + | <U>What we did in the lab:</U></br> | |
| − | <U> Protocol:</U> follow in this link | + | <U>Materials:</U></br> |
| − | </br></br> | + | |
| − | <U>What we did in the lab:</U> | + | |
| − | </br> | + | |
| − | <U>Materials:</U> | + | |
| − | + | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
1. Add all reagent in a 1 ml Eppendorf (1 for C1, 1 for C2 and the last one for pET43.1) </br> | 1. Add all reagent in a 1 ml Eppendorf (1 for C1, 1 for C2 and the last one for pET43.1) </br> | ||
2. Let digest during 1h at 37°C then incubate 5 min at 65°C</br> | 2. Let digest during 1h at 37°C then incubate 5 min at 65°C</br> | ||
| + | For the reagent volumes, refer to the table. </br> | ||
| − | |||
| − | |||
<table> | <table> | ||
<tbody> | <tbody> | ||
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<tr> | <tr> | ||
<td><strong><p> C1/C2= 10 ng/L </p></strong></td> | <td><strong><p> C1/C2= 10 ng/L </p></strong></td> | ||
| − | + | <td> Hind III= 20 000 U/ml </td> | |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
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<figure> | <figure> | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
| − | + | <figcaption> | |
| − | <p><U> Aim:</U> This step is needed to purify our DNA. Indeed, Multiclonal site (parts digested by enzymes but useless) cannot be removed with a PCR clean up because their molecular weight is too low.br> | + | <p> |
| − | + | <U> Aim:</U> This step is needed to purify our DNA. Indeed, Multiclonal site (parts digested by enzymes but useless) cannot be removed with a PCR clean up because their molecular weight is too low.br></br> | |
| + | <U> Protocol:</U> follow in this link</br></br> | ||
| + | <U>What we did in the lab:</U></br> | ||
| + | <U>Materials:</U></br> | ||
| + | <U>Method:</U></br> | ||
| + | 1. 1. Make an 0,7% agarose gel (refer to June 13, 2016) </br> | ||
| + | 2. Fill the electrophoresis chamber with TAEO,5X buffer</br> | ||
| + | 3. Follow the deposit table: </br></br> | ||
| + | Voltmeter: Voltmètre PowerPac HC Biorad</br> | ||
| + | V = 50V</br> | ||
| + | Amp(T=0) = 0.01 A</br></br> | ||
| − | < | + | <table> |
| − | < | + | <thead> |
| − | < | + | <tr> |
| − | < | + | <th></th> |
| − | + | <th>pET43.1a(+)</th> | |
| − | + | <th>C1</th> | |
| − | + | <th>C2</th> | |
| − | + | </tr> | |
| − | + | </thead> | |
| − | + | ||
| − | |||
| − | |||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>pET43.a(+)(ng/µl)</p></strong></td> | ||
| + | <td>23 µl </td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>C1 or C2 10 ng/µl (400 ng)</p></strong></td> | ||
| + | <td></td> | ||
| + | <td>40 µl </td> | ||
| + | <td>40 µl </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>BamHI 20 000 U/ml (10 U)</p></strong></td> | ||
| + | <td>0.5 µl </td> | ||
| + | <td>0.5 µl </td> | ||
| + | <td>0.5 µl </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>HindIII 20 000 U/ml (20 U)</p></strong></td> | ||
| + | <td>1 µl </td> | ||
| + | <td>1 µl </td> | ||
| + | <td>1 V</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>Tampon</p></strong></td> | ||
| + | <td>5 µl </td> | ||
| + | <td>5 µl </td> | ||
| + | <td>5 µl </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>TOTAL</p></strong></td> | ||
| + | <td>50 µl </td> | ||
| + | <td>50 µl </td> | ||
| + | <td>50 µl </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center> Table 12</center></br></br></br> | ||
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| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th> Lines</th> | ||
| + | <th>L1</th> | ||
| + | <th>L2</th> | ||
| + | <th>L3</th> | ||
| + | <th>L4</th> | ||
| + | <th>L5</th> | ||
| + | <th>L6</th> | ||
| + | <th>L7</th> | ||
| + | <th>L8-L9</th> | ||
| + | <th>L10</th> | ||
| + | <th>L11</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| − | < | + | <tbody> |
| − | + | <tr> | |
| − | + | <td><strong><p>Name </p></strong></td> | |
| − | + | <td>Molecular </br> weight</td> | |
| − | + | <td> </td> | |
| − | + | <td>pET43.a(+)</br> uncut</td> | |
| − | + | <td>pET43.1a(+)</br> B</td> | |
| − | + | <td></td> | |
| − | + | <td>pET43.1a(+)</br> H</td> | |
| − | + | <td>pET43.1a(+)</br> B/H</td> | |
| − | + | <td> </td> | |
| − | + | <td> C1</td> | |
| − | + | <td> C2</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td><strong> V(DNA) (µl)</</strong></td> | |
| − | + | <td> 15</td> | |
| − | + | <td></td> | |
| − | + | <td>15</td> | |
| − | + | <td> 15</td> | |
| − | + | <td></td> | |
| − | + | <td> 15</td> | |
| − | + | <td>15</td> | |
| − | + | <td></td> | |
| − | + | <td> 15</td> | |
| − | + | <td>15</td> | |
| − | + | </tr> | |
| − | + | ||
| − | + | ||
| − | + | <tr> | |
| − | + | <td><strong> V(H20) (µl)</</strong></td> | |
| − | + | <td> 0</td> | |
| − | + | <td></td> | |
| − | + | <td>0</td> | |
| − | + | <td> 0</td> | |
| − | + | <td></td> | |
| − | + | <td> 0</td> | |
| − | + | <td>0</td> | |
| − | + | <td></td> | |
| − | + | <td> 0</td> | |
| − | + | <td>0</td> | |
| − | + | </tr> | |
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| − | < | + | <tr> |
| + | <td><strong> V(Load buffer) (µl)</</strong></td> | ||
| + | <td> 3</td> | ||
| + | <td></td> | ||
| + | <td>3</td> | ||
| + | <td> 3</td> | ||
| + | <td></td> | ||
| + | <td> 3</td> | ||
| + | <td>3</td> | ||
| + | <td></td> | ||
| + | <td> 3</td> | ||
| + | <td>3</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table></br> | ||
| + | <center>Table 13</center></br></br> | ||
| + | <U>Results:</U></br> | ||
| − | <center><img src=" https://static.igem.org/mediawiki/2016/0/04/4._Fig3_Pasteur.png" width="300px"; alt=""></center> | + | |
| − | <center>Figure 3</center> | + | <center><img src=" https://static.igem.org/mediawiki/2016/0/04/4._Fig3_Pasteur.png" width="300px"; alt=""></center> |
| − | </p> | + | <center>Figure 3</center> |
| − | </figcaption> | + | </p> |
| − | + | </figcaption> | |
| + | </figure> | ||
</div> | </div> | ||
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<figure> | <figure> | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
| − | + | <figcaption> | |
| − | <p ><U> Aim:</U> We want to take back the plasmid and the inserts digested. </br> | + | <p> |
| − | + | <U> Aim:</U> We want to take back the plasmid and the inserts digested. </br> | |
| + | The electrophoresis has set up the waste and the parts needed</br> | ||
| + | <U> Protocol:</U> follow in this link</br></br> | ||
| + | <U>What we did in the lab:</U></br> | ||
| + | <U>Materials:</U></br> | ||
| + | <U>Materials:</U></br> | ||
| + | <U>Method:</U></br> | ||
| + | 1. Cut agarose gel to have only parts of plasmid that we need and put it on an 1ml Eppendorf</br> | ||
| − | |||
| − | |||
| − | |||
| − | |||
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| − | < | + | <table> |
| − | + | <thead> | |
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>Empty eppendorf (g)</th> | ||
| + | <th>Eppendorf + gel (g)</th> | ||
| + | <th>Final weight (mg)</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| − | < | + | <tbody> |
| − | + | <tr> | |
| − | + | <td><strong><p>m (pET43.1a(+)</p></strong></td> | |
| − | + | <td>0.99</td> | |
| − | + | <td>1.12 </td> | |
| − | + | <td>127.2</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td><strong> m(C1)</strong></td> | |
| − | + | <td> 0.99</td> | |
| − | + | <td>1.07</td> | |
| − | + | <td>76.5</td> | |
| − | + | </tr> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | <tr> | ||
| + | <td><strong> m(C2)</strong></td> | ||
| + | <td> 0.99</td> | ||
| + | <td>1.09</td> | ||
| + | <td>106.1</td> | ||
| + | </tr> | ||
| − | + | </tbody> | |
| + | </table> | ||
| + | </br> | ||
| + | <center>Table 13</center></br></br> | ||
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| − | |||
| − | |||
| − | |||
| − | |||
| − | < | + | 2. We use NEB extraction kit: De Macherey-Nagel_nucleospin-Gel and PCR Clean-up</br></br> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | < | + | For NT1 volumes: we need 200L of NT1 for 100 mg DNA</br> |
| + | For pET43.1 volume = 254 L of NT1 buffer with p1000</br> | ||
| + | For C1 volumes= 153L of NT1 buffer with p1000</br> | ||
| + | For C2 volumes= 212L of NT1 buffer with p1000</br> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | |||
| − | < | + | <div class="lightbox" id="exp9"> |
| − | < | + | <figure> |
| − | < | + | <a href="#" class="closemsg"></a> |
| − | < | + | <figcaption> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | <U>Method:</U></br> | + | <p> |
| − | Refer to June 16, 2016 </br> | + | <U> Aim:</U> After the digestion we dephosphorylate It to prevent that it binds without the insert. </br> </br> |
| − | C(pET43.1) = 500 ng pour 20L</br> | + | <U> Protocol:</U> follow in this link</br></br> |
| − | C(C1/C2) = 400 ng pour 20L</br> | + | <U>What we did in the lab:</U></br> |
| − | </p> | + | <U>Materials:</U></br> |
| − | </figcaption> | + | <U>Materials:</U> |
| − | + | <U>Method:</U></br> | |
| + | Refer to June 16, 2016 </br> | ||
| + | C(pET43.1) = 500 ng pour 20L</br> | ||
| + | C(C1/C2) = 400 ng pour 20L</br> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
<div class="lightbox" id="exp10"> | <div class="lightbox" id="exp10"> | ||
<figure> | <figure> | ||
| − | + | <a href="#" class="closemsg"></a> | |
| − | + | <figcaption> | |
| − | <p><U> Aim:</U> Let the pET43.1 to bind with the insert.</br> </br> | + | <p> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <U> Aim:</U> Let the pET43.1 to bind with the insert.</br> </br> |
| − | <U>What we did in the lab:</U></br> | + | <U> Protocol:</U> follow in this link</br></br> |
| − | <U>Materials:</U></br> | + | <U>What we did in the lab:</U></br> |
| − | <U>Method:</U></br> | + | <U>Materials:</U></br> |
| − | 1. Add all reagents in a 1ml Eppendorf for each sample. Follow the table. | + | <U>Method:</U></br> |
| − | We have to do the ligation in the smallest volume to have more efficiency.S | + | 1. Add all reagents in a 1ml Eppendorf for each sample. Follow the table. |
| + | We have to do the ligation in the smallest volume to have more efficiency.S | ||
| − | <table> | + | <table> |
| − | + | <thead> | |
| − | + | <tr> | |
| − | + | <th></th> | |
| − | + | <th>1 :1 </br> Lab Method</th> | |
| − | + | <th>1 : 3 </br> Lab Method</th> | |
| − | + | <th>Only Pet43.1a(+) </br> Lab Method</th> | |
| − | + | <th>1 :1 </b>Academic Method</th> | |
| − | + | <th>1 :3 </br> Academic Method</th> | |
| − | + | </tr> | |
| − | + | </thead> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <tr> | + | <tbody> |
| − | + | <tr> | |
| − | + | <td><strong><p>pET43.1a(+) (50 mg)</p></strong></td> | |
| − | + | <td>2 μl</td> | |
| − | + | <td>2 μl</td> | |
| − | + | <td>2 μl</td> | |
| − | + | <td>2 μl</td> | |
| − | </tr> | + | <td>2 μl</td> |
| − | + | </tr> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | <tr> | ||
| + | <td><strong> <p>Insert C1-C2</p></strong></td> | ||
| + | <td>2.5 μl</td> | ||
| + | <td>7.5 μl</td> | ||
| + | <td></td> | ||
| + | <td>2.5 μl</td> | ||
| + | <td>7.5 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong> <p>T4 ligase</p></strong></td> | ||
| + | <td>1 μl</td> | ||
| + | <td>1 μl</td> | ||
| + | <td>1 μl</td> | ||
| + | <td>2 μl</td> | ||
| + | <td>2 μl</td> | ||
| + | </tr> | ||
| − | </p> | + | <tr> |
| − | </figcaption> | + | <td><strong> <p>Tampon 10X </p></strong></td> |
| − | + | <td>2 μl</td> | |
| + | <td>2 μl</td> | ||
| + | <td>2 μl</td> | ||
| + | <td>2 μl</td> | ||
| + | <td>2 μl</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong> <p>H20</p></strong></td> | ||
| + | <td>12.5 μl</td> | ||
| + | <td>8.5 μl</td> | ||
| + | <td>15 μl</td> | ||
| + | <td>12.3 μl</td> | ||
| + | <td>7.3 μl</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td>20 μl</td> | ||
| + | <td>20 μl</td> | ||
| + | <td>20 μl</td> | ||
| + | <td>20 μl</td> | ||
| + | <td>20 μl</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table></br> | ||
| + | <center>Table 15</center></br></br> | ||
| + | 2. Let ligate 10 min at 37°C then incubate 10 min at 65°C to inactivate the enzymes</br> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| Line 825: | Line 828: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)</br></br> | + | <p> |
| + | <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> | ||
| − | + | • Nanodrop (Thermofisher)</br> | |
| − | + | • Elution buffer from QIAGEN kit</br> | |
| − | + | • Microbiology equipment (Follow this link)</br></br> | |
| − | </br> | + | |
| − | + | ||
| − | + | ||
| − | + | <U>Method:</U></br> | |
| − | + | Analyze absorbance at 260nm</br> | |
| − | + | 1. Clean the Nanodrop with water</br> | |
| + | 2. Make the blank with 1 µl of elution buffer</br> | ||
| + | 3. Put 1ul of your sample on the Nanodrop</br> | ||
| + | 4. Make the measure and clean the Nanodrop between each measure</br></br> | ||
| + | <U>Results:</U></br> | ||
| − | < | + | <table> |
| − | + | <thead> | |
| − | 1 | + | <tr> |
| − | 2 | + | <th>λ= 260 nm</th> |
| − | + | <th>B1(1)</th> | |
| − | + | <th>B1(2)</th> | |
| + | <th>C2>th> | ||
| + | </tr> | ||
| + | </thead> | ||
| − | + | <tbody> | |
| − | + | <tr> | |
| − | + | <td><strong><p>A<sub>DNA</sub></p></strong></td> | |
| − | + | <td>0.725</td> | |
| − | + | <td>0.741</td> | |
| − | + | <td>0.761</td> | |
| − | + | </tr> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | 5. Preparation of the samples for sequencing: put 15 µl of DNA and add 2 µl of fitted primers</br> | + | <tr> |
| − | 6. Send the samples for sequencing</br> | + | <tr> |
| − | </p> | + | <td><strong>C final</strong></td> |
| − | </figcaption> | + | <td>36.3 ng/µl</td> |
| + | <td>37.0 ng/µl</td> | ||
| + | <td>38.0 ngµl</td> | ||
| + | </tr> | ||
| + | |||
| + | </tbody> | ||
| + | </table> | ||
| + | <U>Absorbance</U> | ||
| + | </br></br></br> | ||
| + | |||
| + | 5. Preparation of the samples for sequencing: put 15 µl of DNA and add 2 µl of fitted primers</br> | ||
| + | 6. Send the samples for sequencing</br> | ||
| + | </p> | ||
| + | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| Line 887: | Line 890: | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> : Enter the plasmid into the bacteria </br> </br> | + | <p> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <U> Aim:</U> : Enter the plasmid into the bacteria </br> </br> |
| − | <U>What we did in the lab:</U></br> | + | <U> Protocol:</U> follow in this link</br></br> |
| − | refer to June 6, 2016 but with these quantities: </br> | + | <U>What we did in the lab:</U></br> |
| − | - L of bacteria</br> | + | refer to June 6, 2016 but with these quantities: </br> |
| − | - L of DNA</br> | + | - L of bacteria</br> |
| − | - L of SOC</br></br> | + | - L of DNA</br> |
| − | Spread each sample on a petri dish made of LB and CB50 (carbenicillin 50g/ml) </br> | + | - L of SOC</br></br> |
| − | </p> | + | Spread each sample on a petri dish made of LB and CB50 (carbenicillin 50g/ml) </br> |
| − | </figcaption> | + | </p> |
| + | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| Line 906: | Line 910: | ||
<figure> | <figure> | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
| − | + | <figcaption> | |
| − | <p><U> Aim:</U> We want to assay DNA of C1 and C2 but we do not have enough insert so we tried different dilutions.</br>.</br> | + | <p> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <U> Aim:</U> We want to assay DNA of C1 and C2 but we do not have enough insert so we tried different dilutions.</br>.</br> |
| − | <U>What we did in the lab:</U></br> | + | <U> Protocol:</U> follow in this link</br></br> |
| − | <U>Method:</U></br> | + | <U>What we did in the lab:</U></br> |
| − | 1. first, we use 1L of C1 and 49L of water</br> | + | <U>Method:</U></br> |
| − | 2. we made the blank with H20</br> | + | 1. first, we use 1L of C1 and 49L of water</br> |
| − | 3. we use the spectrophotometer utrospec 3100 pro_amersham Bioscience at =260 nm</br> | + | 2. we made the blank with H20</br> |
| − | 4. the results were not convincing so we decided to use a Nanodrop</br> | + | 3. we use the spectrophotometer utrospec 3100 pro_amersham Bioscience at =260 nm</br> |
| + | 4. the results were not convincing so we decided to use a Nanodrop</br> | ||
| + | <U>Results:</U></br> | ||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>>λ= 260 nm </th> | ||
| + | <th>C1</th> | ||
| + | <th>C2</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| − | + | <tbody> | |
| − | + | <tr> | |
| − | + | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> | |
| − | + | <td>1.71</td> | |
| − | + | <td>1.8</td> | |
| − | + | </tr> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | </p> | + | <tr> |
| − | </figcaption> | + | <td><strong><p>A<span>260</span> /A<span>230</span> </p></strong></td> |
| + | <td>0.39</td> | ||
| + | <td>0.39</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>Cfinal </p></strong></td> | ||
| + | <td>9.7 ng/μl</td> | ||
| + | <td> 11 ng/μl </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | |||
| + | </table> | ||
| + | <center> Table 16</center></br></br></br> | ||
| + | |||
| + | </p> | ||
| + | </figcaption> | ||
</figure> | </figure> | ||
| − | |||
| − | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 18:26, 30 September 2016
