Difference between revisions of "Team:Pasteur Paris/Microbiology week3"
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<div> | <div> | ||
| + | <h2><B>Microbiology Notebook</B></h2> | ||
| + | </div> | ||
| − | + | <div id="home"> | |
| + | <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> | ||
| + | </div> | ||
<div id="week3"> | <div id="week3"> | ||
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<p><h3><B> June 20, 2016:</B></h3></p> | <p><h3><B> June 20, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp1"><h4> Dosage of pET43.1</h4></a></br> | + | <a href="#exp1"><h4> 16. Dosage of pET43.1</h4></a></br> |
| − | <a href="#exp2"><h4> Checkin of stab cultures</h4></a></br> | + | <a href="#exp2"><h4> 17. Checkin of stab cultures</h4></a></br> |
| − | <a href="#exp3"><h4> Transformation of pET43.1a (+)</h4></a></br> | + | <a href="#exp3"><h4> 18. Transformation of pET43.1a (+)</h4></a></br> |
</p> | </p> | ||
<p><h3><B> June 21, 2016:</B></h3></p> | <p><h3><B> June 21, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp4"><h4> Analyse </h4></a></br> | + | <a href="#exp4"><h4> 19. Analyse </h4></a></br> |
| − | <a href="#exp5"><h4> Dosage of pET43.1a(+) </h4></a></br> | + | <a href="#exp5"><h4> 20. Dosage of pET43.1a(+) </h4></a></br> |
| − | <a href="#exp6"><h4> Digestion of pET43.1 and C1 and C2</h4></a></br> | + | <a href="#exp6"><h4> 21. Digestion of pET43.1 and C1 and C2</h4></a></br> |
| − | <a href="#exp7"><h4> Electrophoresis of pET43.1a(+) and the inserts C1 and | + | <a href="#exp7"><h4> 22. Electrophoresis of pET43.1a(+) and the inserts C1 and C:</h4></a></br> |
| − | <a href="#exp8"><h4> Gel extraction | + | <a href="#exp8"><h4> 23. Gel extraction</h4></a></br> |
| − | <a href="#exp9"><h4> Dephosphorylation of pET43.1a(+) | + | <a href="#exp9"><h4> 24. Dephosphorylation of pET43.1a(+)</h4></a></br> |
</p> | </p> | ||
<p><h3><B> June 22, 2016:</B></h3></p> | <p><h3><B> June 22, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp10"><h4> Ligation of pET43.1a(+) with C1 and C2 </h4></a></br> | + | <a href="#exp10"><h4> 25. 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> | + | <a href="#exp11"><h4> 26. Transformation of pET43.1a(+) linked with C1 and C2</h4></a></br> |
</p> | </p> | ||
<p><B><h3> June 23, 2016:</B></h3></p> | <p><B><h3> June 23, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp12"><h4 Dosage of the uncut inserts</h4></a></br> | + | <a href="#exp12"><h4> 27. Dosage of the uncut inserts</h4></a></br> |
</p> | </p> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp1"> |
<figure> | <figure> | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <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> | + | <h6><U> Aim:</U></h6> 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><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> 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> | ||
| Line 261: | Line 275: | ||
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></ | + | <h6><U> Results: </U></h6> |
| − | C1=87 ng/ | + | C1=87 ng/l</br> |
| − | C2 = 103.3 ng/ | + | 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> | ||
| − | C1=0,2 ng/ | + | C1=0,2 ng/l</br> |
| − | C2= 0,8 ng/ | + | C2= 0,8 ng/l</br></br> |
| − | 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><br /> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 10</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 284: | Line 299: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<sub>260</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<sub>260</sub></p></strong></td> |
| − | <td>0.043 </td> | + | <td align = "center"; valign = "center">0.043 </td> |
| − | <td>0.59 </td> | + | <td align = "center"; valign = "center">0.59 </td> |
| − | <td>0.037 </td> | + | <td align = "center"; valign = "center">0.037 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<sub>280</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<sub>280</sub></p></strong></td> |
| − | <td>0.026 </td> | + | <td align = "center"; valign = "center">0.026 </td> |
| − | <td>0.01 </td> | + | <td align = "center"; valign = "center">0.01 </td> |
| − | <td>0.016 </td> | + | <td align = "center"; valign = "center">0.016 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> |
| − | <td>1.7 </td> | + | <td align = "center"; valign = "center">1.7 </td> |
| − | <td>5.9 </td> | + | <td align = "center"; valign = "center">5.9 </td> |
| − | <td>2.31 </td> | + | <td align = "center"; valign = "center">2.31 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> |
| − | <td>2.15 </td> | + | <td align = "center"; valign = "center">2.15 </td> |
| − | <td>1.95 </td> | + | <td align = "center"; valign = "center">1.95 </td> |
| − | <td>1.85 </td> | + | <td align = "center"; valign = "center">1.85 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> |
| − | <td>143.33 </td> | + | <td align = "center"; valign = "center">143.33 </td> |
| − | <td>130 </td> | + | <td align = "center"; valign = "center">130 </td> |
| − | <td>123.33 </td> | + | <td align = "center"; valign = "center">123.33 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> |
| − | <td>133.22 </td> | + | <td align = "center"; valign = "center">133.22 </td> |
| − | <td>133.22 </td> | + | <td align = "center"; valign = "center">133.22 </td> |
| − | <td>133.22 </td> | + | <td align = "center"; valign = "center">133.22 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></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).</br></br> | + | Then, we resuspend the insert by adding buffer TE1X in each tube (refer to the June 15, 2016 for the volumes). <br /> |
| + | </br></br><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 341: | Line 356: | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Results:</U> Colonies on the petri dish were counted. We saw bacterial colonies so the stab culture was successful. </br> </br> | + | <p> |
| + | <h6><U> Results:</U></h6> Colonies on the petri dish were counted. We saw bacterial colonies so the stab culture was successful. </br> | ||
| + | </br><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
| Line 351: | Line 370: | ||
<a href="#" class="closemsg"></a> | <a href="#" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Refer to June 16, 2016.</br> </br> | + | <p> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Aim:</U></h6> Refer to June 16, 2016.</br></br> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br><br /> |
| + | <h6><U>What we did in the lab:</U></h6></br> | ||
Refer to June 16, 2016, for protocol (dephosphorylation, ligation with C1 and C2 and transformation). </br> | Refer to June 16, 2016, for protocol (dephosphorylation, ligation with C1 and C2 and transformation). </br> | ||
| − | We spread the mix in petri dish with CB50 and CM34 to see if the transformation was successful. | + | We spread the mix in petri dish with CB50 and CM34 to see if the transformation was successful.<br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp4"> | <div class="lightbox" id="exp4"> | ||
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<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.</br> </br> | + | <p> |
| + | <h6><U> Results:</U></h6> 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><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> To link pET43.1a(+) with C1 and C2 we assayed before plasmid DNA but perhaps our ligation doesn’t work. We want to know the concentration of our samples. </br> </br> | + | <h6><U> Aim:</U></h6> To link pET43.1a(+) with C1 and C2 we assayed before plasmid DNA but perhaps our ligation doesn’t work. We want to know the concentration of our samples. </br> </br> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. We need more buffer solution so we dilute TE 10X by ten to have TE. </br> | 1. We need more buffer solution so we dilute TE 10X by ten to have TE. </br> | ||
In a 250 ml burette, we had 25ml of TE and we complete with water until 250 ml. We aliquot the 250 ml in fixe 50 ml bottle. </br> | In a 250 ml burette, we had 25ml of TE and we complete with water until 250 ml. We aliquot the 250 ml in fixe 50 ml bottle. </br> | ||
| − | 2. We made a 15/1000 dilutions with our samples (15 | + | 2. We made a 15/1000 dilutions with our samples (15 l of DNA and 985 l of water) </br> |
| − | 3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back | + | 3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back 15 l and add of DNA</br> |
| − | 4. We analyse at 260 nm | + | 4. We analyse at 260 nm</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 11</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| − | <th | + | <th>λ= 260 nm </th> |
<th>Sample 1</th> | <th>Sample 1</th> | ||
<th>Sample 2</th> | <th>Sample 2</th> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<sub>260</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<sub>260</sub></p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> A<sub>280</sub> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> A<sub>280</sub> </p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td | + | <td align = "center"; valign = "center"> </td> |
| − | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>dilué</span>(ng/µl)</sub></p></strong></td> |
| − | <td>1.25</td> | + | <td align = "center"; valign = "center">1.25</td> |
| − | <td>1 </td> | + | <td align = "center"; valign = "center">1 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>final</span>(ng/µl)</sub></p></strong></td> |
| − | <td>250 </td> | + | <td align = "center"; valign = "center">250 </td> |
| − | <td>200 </td> | + | <td align = "center"; valign = "center">200 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C<sub><span>medium</span>(ng/µl)</sub></p></strong></td> |
| − | <td>225 </td> | + | <td align = "center"; valign = "center">225 </td> |
| − | <td>225 </td> | + | <td align = "center"; valign = "center">225 </td> |
| − | <td>225 </td> | + | <td align = "center"; valign = "center">225 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 452: | Line 477: | ||
<figcaption> | <figcaption> | ||
<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> | + | <h6><U> Aim:</U></h6> 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> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
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> | + | For the reagent volumes, refer to the table. </br><br /> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 12</U></p></i></caption> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p> pET43.1= 130 ng/L</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> pET43.1= 130 ng/L</p></strong></td> |
| − | <td> BamHI= 20 000 U/ml </td> | + | <td align = "center"; valign = "center"> BamHI= 20 000 U/ml </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> C1/C2= 10 ng/L </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p> C1/C2= 10 ng/L </p></strong></td> |
| − | <td> Hind III= 20 000 U/ml </td> | + | <td align = "center"; valign = "center"> Hind III= 20 000 U/ml </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /> |
| − | + | </br></br></br> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 487: | Line 513: | ||
<p> | <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> | + | <h6><U> Aim:</U></h6> 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> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | + | 1. Make an 0,7% agarose gel (refer to June 13, 2016) </br> | |
2. Fill the electrophoresis chamber with TAEO,5X buffer</br> | 2. Fill the electrophoresis chamber with TAEO,5X buffer</br> | ||
3. Follow the deposit table: </br></br> | 3. Follow the deposit table: </br></br> | ||
| Line 500: | Line 526: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 13</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 512: | Line 539: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.a(+)(ng/µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.a(+)(ng/µl)</p></strong></td> |
| − | <td>23 µl </td> | + | <td align = "center"; valign = "center">23 µl </td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>C1 or C2 10 ng/µl (400 ng)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>C1 or C2 10 ng/µl (400 ng)</p></strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>40 µl </td> | + | <td align = "center"; valign = "center">40 µl </td> |
| − | <td>40 µl </td> | + | <td align = "center"; valign = "center">40 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>BamHI 20 000 U/ml (10 U)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>BamHI 20 000 U/ml (10 U)</p></strong></td> |
| − | <td>0.5 µl </td> | + | <td align = "center"; valign = "center">0.5 µl </td> |
| − | <td>0.5 µl </td> | + | <td align = "center"; valign = "center">0.5 µl </td> |
| − | <td>0.5 µl </td> | + | <td align = "center"; valign = "center">0.5 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>HindIII 20 000 U/ml (20 U)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>HindIII 20 000 U/ml (20 U)</p></strong></td> |
| − | <td>1 µl </td> | + | <td align = "center"; valign = "center">1 µl </td> |
| − | <td>1 µl </td> | + | <td align = "center"; valign = "center">1 µl </td> |
| − | <td>1 V</td> | + | <td align = "center"; valign = "center">1 V</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Tampon</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Tampon</p></strong></td> |
| − | <td>5 µl </td> | + | <td align = "center"; valign = "center">5 µl </td> |
| − | <td>5 µl </td> | + | <td align = "center"; valign = "center">5 µl </td> |
| − | <td>5 µl </td> | + | <td align = "center"; valign = "center">5 µl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>TOTAL</p></strong></td> |
| − | <td>50 µl </td> | + | <td align = "center"; valign = "center">50 µl </td> |
| − | <td>50 µl </td> | + | <td align = "center"; valign = "center">50 µl </td> |
| − | <td>50 µl </td> | + | <td align = "center"; valign = "center">50 µl </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | + | ||
| − | + | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 14</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 579: | Line 604: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name </p></strong></td> |
| − | <td>Molecular </br> weight</td> | + | <td align = "center"; valign = "center">Molecular </br> weight</td> |
| − | <td> </td> | + | <td align = "center"; valign = "center"> </td> |
| − | <td>pET43.a(+)</br> uncut</td> | + | <td align = "center"; valign = "center">pET43.a(+)</br> uncut</td> |
| − | <td>pET43.1a(+)</br> B</td> | + | <td align = "center"; valign = "center">pET43.1a(+)</br> B</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+)</br> H</td> | + | <td align = "center"; valign = "center">pET43.1a(+)</br> H</td> |
| − | <td>pET43.1a(+)</br> B/H</td> | + | <td align = "center"; valign = "center">pET43.1a(+)</br> B/H</td> |
| − | <td> </td> | + | <td align = "center"; valign = "center"> </td> |
| − | <td> C1</td> | + | <td align = "center"; valign = "center"> C1</td> |
| − | <td> C2</td> | + | <td align = "center"; valign = "center"> C2</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> V(DNA) (µl)</</strong></td> | + | <td align = "center"; valign = "center"><strong> V(DNA) (µl)</</strong></td> |
| − | <td> 15</td> | + | <td align = "center"; valign = "center"> 15</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td> 15</td> | + | <td align = "center"; valign = "center"> 15</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 15</td> | + | <td align = "center"; valign = "center"> 15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 15</td> | + | <td align = "center"; valign = "center"> 15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> V(H20) (µl)</</strong></td> | + | <td align = "center"; valign = "center"><strong> V(H20) (µl)</</strong></td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 0</td> | + | <td align = "center"; valign = "center"> 0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> V(Load buffer) (µl)</</strong></td> | + | <td align = "center"; valign = "center"><strong> V(Load buffer) (µl)</</strong></td> |
| − | <td> 3</td> | + | <td align = "center"; valign = "center"> 3</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>3</td> | + | <td align = "center"; valign = "center">3</td> |
| − | <td> 3</td> | + | <td align = "center"; valign = "center"> 3</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 3</td> | + | <td align = "center"; valign = "center"> 3</td> |
| − | <td>3</td> | + | <td align = "center"; valign = "center">3</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> 3</td> | + | <td align = "center"; valign = "center"> 3</td> |
| − | <td>3</td> | + | <td align = "center"; valign = "center">3</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table></br> | + | </table></br><br /> |
| − | + | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6> |
| − | <center><img src=" https://static.igem.org/mediawiki/2016/0/04/4._Fig3_Pasteur.png" width="300px"; alt=""></center> | + | <center><img src=" https://static.igem.org/mediawiki/2016/0/04/4._Fig3_Pasteur.png" width="300px"; alt=""><i><p><U>Figure 1</U></p></i></center><br /> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 655: | Line 679: | ||
<p> | <p> | ||
| − | <U> Aim:</U> We want to take back the plasmid and the inserts digested. </br> | + | <h6><U> Aim:</U></h6> We want to take back the plasmid and the inserts digested. </br> |
| − | The electrophoresis has set up the waste and the parts needed</br> | + | The electrophoresis has set up the waste and the parts needed</br><br /> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | < | + | <h6><U>Method:</U></h6> |
| − | + | 1. Cut agarose gel to have only parts of plasmid that we need and put it on an 1ml Eppendorf</br><br /> | |
| − | 1. Cut agarose gel to have only parts of plasmid that we need and put it on an 1ml Eppendorf</br> | + | |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 15</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 678: | Line 702: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>m (pET43.1a(+)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>m (pET43.1a(+)</p></strong></td> |
| − | <td>0.99</td> | + | <td align = "center"; valign = "center">0.99</td> |
| − | <td>1.12 </td> | + | <td align = "center"; valign = "center">1.12 </td> |
| − | <td>127.2</td> | + | <td align = "center"; valign = "center">127.2</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> m(C1)</strong></td> | + | <td align = "center"; valign = "center"><strong> m(C1)</strong></td> |
| − | <td> 0.99</td> | + | <td align = "center"; valign = "center"> 0.99</td> |
| − | <td>1.07</td> | + | <td align = "center"; valign = "center">1.07</td> |
| − | <td>76.5</td> | + | <td align = "center"; valign = "center">76.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> m(C2)</strong></td> | + | <td align = "center"; valign = "center"><strong> m(C2)</strong></td> |
| − | <td> 0.99</td> | + | <td align = "center"; valign = "center"> 0.99</td> |
| − | <td>1.09</td> | + | <td align = "center"; valign = "center">1.09</td> |
| − | <td>106.1</td> | + | <td align = "center"; valign = "center">106.1</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | + | ||
| Line 707: | Line 729: | ||
For NT1 volumes: we need 200L of NT1 for 100 mg DNA</br> | For NT1 volumes: we need 200L of NT1 for 100 mg DNA</br> | ||
| − | For pET43.1 volume = 254 | + | For pET43.1 volume = 254 l of NT1 buffer with p1000</br> |
| − | For C1 volumes= | + | For C1 volumes= 153 l of NT1 buffer with p1000</br> |
| − | For C2 volumes= | + | For C2 volumes= 212 l of NT1 buffer with p1000</br> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 722: | Line 745: | ||
<p> | <p> | ||
| − | <U> Aim:</U> After the digestion we dephosphorylate It to prevent that it binds without the insert. </br> </br> | + | <h6><U> Aim:</U></h6> After the digestion we dephosphorylate It to prevent that it binds without the insert. </br></br> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | ||
| − | + | <h6><U>Method:</U></h6> | |
| − | + | ||
Refer to June 16, 2016 </br> | Refer to June 16, 2016 </br> | ||
| − | C(pET43.1) = 500 ng pour | + | C(pET43.1) = 500 ng pour 20 l</br> |
| − | C(C1/C2) = 400 ng pour | + | C(C1/C2) = 400 ng pour 20 l</br> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp10"> | <div class="lightbox" id="exp10"> | ||
| Line 741: | Line 766: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Let the pET43.1 to bind with the insert.</br> </br> | + | <h6><U> Aim:</U></h6> Let the pET43.1 to bind with the insert.</br> </br> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></ | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | + | <h6><U>Method:</U></h6> | |
| − | <U>Method:</U></ | + | 1. Add all reagents in a 1ml Eppendorf for each sample. Follow the table. |
| − | 1. | + | |
We have to do the ligation in the smallest volume to have more efficiency.S | We have to do the ligation in the smallest volume to have more efficiency.S | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 16</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 763: | Line 788: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+) (50 mg)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+) (50 mg)</p></strong></td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Insert C1-C2</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Insert C1-C2</p></strong></td> |
| − | <td>2.5 μl</td> | + | <td align = "center"; valign = "center">2.5 μl</td> |
| − | <td>7.5 μl</td> | + | <td align = "center"; valign = "center">7.5 μl</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2.5 μl</td> | + | <td align = "center"; valign = "center">2.5 μl</td> |
| − | <td>7.5 μl</td> | + | <td align = "center"; valign = "center">7.5 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>T4 ligase</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>T4 ligase</p></strong></td> |
| − | <td>1 μl</td> | + | <td align = "center"; valign = "center">1 μl</td> |
| − | <td>1 μl</td> | + | <td align = "center"; valign = "center">1 μl</td> |
| − | <td>1 μl</td> | + | <td align = "center"; valign = "center">1 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>Tampon 10X </p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>Tampon 10X </p></strong></td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
| − | <td>2 μl</td> | + | <td align = "center"; valign = "center">2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> <p>H20</p></strong></td> | + | <td align = "center"; valign = "center"><strong> <p>H20</p></strong></td> |
| − | <td>12.5 μl</td> | + | <td align = "center"; valign = "center">12.5 μl</td> |
| − | <td>8.5 μl</td> | + | <td align = "center"; valign = "center">8.5 μl</td> |
| − | <td>15 μl</td> | + | <td align = "center"; valign = "center">15 μl</td> |
| − | <td>12.3 μl</td> | + | <td align = "center"; valign = "center">12.3 μl</td> |
| − | <td>7.3 μl</td> | + | <td align = "center"; valign = "center">7.3 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td>20 μl</td> | + | <td align = "center"; valign = "center">20 μl</td> |
| − | <td>20 μl</td> | + | <td align = "center"; valign = "center">20 μl</td> |
| − | <td>20 μl</td> | + | <td align = "center"; valign = "center">20 μl</td> |
| − | <td>20 μl</td> | + | <td align = "center"; valign = "center">20 μl</td> |
| − | <td>20 μl</td> | + | <td align = "center"; valign = "center">20 μl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
2. Let ligate 10 min at 37°C then incubate 10 min at 65°C to inactivate the enzymes</br> | 2. Let ligate 10 min at 37°C then incubate 10 min at 65°C to inactivate the enzymes</br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</div> | </div> | ||
| Line 891: | Line 855: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> : Enter the plasmid into the bacteria </br> </br> | + | <h6><U> Aim:</U></h6> : Enter the plasmid into the bacteria </br> </br> |
| − | <U> Protocol:</U> follow in this link</br></br> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
refer to June 6, 2016 but with these quantities: </br> | refer to June 6, 2016 but with these quantities: </br> | ||
- L of bacteria</br> | - L of bacteria</br> | ||
| Line 913: | Line 877: | ||
<p> | <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> | + | <h6><U> Aim:</U></h6> We want to assay DNA of C1 and C2 but we do not have enough insert so we tried different dilutions.</br></br><br /> |
| − | + | <h6><U>What we did in the lab:</U></h6></br> | |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>Method:</U></h6> |
| − | <U>Method:</U></ | + | 1. First, we use 1 μl of C1 and 49 μl of water.</br> |
| − | 1. | + | 2. Make the blank with H20.</br> |
| − | 2. | + | 3. Use the spectrophotometer Utrospec 3100 pro_amersham Bioscience at 260 nm</br> |
| − | 3. | + | 4. Results were not convincing so we decided to use a Nanodrop</br></br> |
| − | 4. | + | |
| − | <U>Results:</U></br> | + | <h6><U>Results:</U></h6></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p><U>Table 17</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 933: | Line 898: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260</span> /A<span>280</span> </p></strong></td> |
| − | <td>1.71</td> | + | <td align = "center"; valign = "center">1.71</td> |
| − | <td>1.8</td> | + | <td align = "center"; valign = "center">1.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>A<span>260</span> /A<span>230</span> </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>A<span>260</span> /A<span>230</span> </p></strong></td> |
<td>0.39</td> | <td>0.39</td> | ||
| − | <td>0.39</td> | + | <td align = "center"; valign = "center">0.39</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinal </p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinal </p></strong></td> |
| − | <td>9.7 ng/μl</td> | + | <td align = "center"; valign = "center">9.7 ng/μl</td> |
| − | <td> 11 ng/μl </td> | + | <td align = "center"; valign = "center"> 11 ng/μl </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | </br></br></br> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
| − | |||
</div> | </div> | ||
| + | |||
</body> | </body> | ||
| − | |||
</html> | </html> | ||
