Difference between revisions of "Team:Pasteur Paris/Microbiology week5"
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<div> | <div> | ||
| Line 226: | Line 237: | ||
</div> | </div> | ||
<div id="week5"> | <div id="week5"> | ||
| − | <p><h5><B>Week 5</B></h5></p> | + | <p><h5><B>Week 5</B></h5></p> |
| − | <p><h3><B>July 4, 2016:</B></h3></p> | + | <p><h3><B>July 4, 2016:</B></h3></p> |
<p> | <p> | ||
<a href="#exp1"><h4> 47.Digestion of pET43.1 (a) with XbaI and HindIII </h4></a></br> | <a href="#exp1"><h4> 47.Digestion of pET43.1 (a) with XbaI and HindIII </h4></a></br> | ||
<a href="#exp2"><h4> 48. DNA measurements</h4></a></br> | <a href="#exp2"><h4> 48. DNA measurements</h4></a></br> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | <p><h3><B>July 5, 2016: </B></h3></p> | + | <a href="#exp3"><h4> 50. Electrophoresis on agarose gel of pET43.1a(+) digest by XbaI and HindIII</h4></a></br> |
| + | <a href="#exp4"><h4> 51. Extraction gel of pET43.1a(+) digest by XbaI and HindIII</h4></a></br> | ||
| + | <a href="#exp5"><h4> 52. Dephosphorylation of pET43.1a(+) (digest by XbaI/HindIII) done previously</h4></a></br> | ||
| + | </p> | ||
| + | <p><h3><B>July 5, 2016: </B></h3></p> | ||
<p> | <p> | ||
| − | <a href="# | + | <a href="#exp6"><h4> 53. Dephosphorylation of pET43.1a(+)digest by XbaI/HindIII) </h4></a></br> |
| − | <a href="# | + | <a href="#exp7"><h4> 54. Ligation of dephosphorylated pET43.1a(+) with C2</h4></a></br> |
| − | <a href="# | + | <a href="#exp8"><h4> 55. Ligation of pET43.1a(+) dephosphorylate with C2</h4></a></br> |
| − | <a href="# | + | <a href="#exp9"><h4> 56. Electrophoresis on agarose gel of pET43.1 digest by XbaI and HindIII</h4></a></br> |
| − | <a href="# | + | <a href="#exp10"><h4> 57. Electrophoresis of pET43.1a(+) digested by XbaI and Hind III </h4></a></br> |
| − | <a href="# | + | <a href="#exp11"><h4> 58. Plasmid concentrations </h4></a></br> |
| − | <a href="# | + | <a href="#exp12"><h4> 59. Transformation of DH5 competent cells </h4></a></br> |
| − | + | ||
</p> | </p> | ||
| − | <p><h3><B>July 6, 2016: </B></h3></p> | + | <p><h3><B>July 6, 2016: </B></h3></p> |
<p> | <p> | ||
| − | <a href="# | + | <a href="#exp13"><h4> 60. Verification of transformation of the 5/07/16</h4></a></br> |
| − | <a href="# | + | <a href="#exp14"><h4>61. Cultivating colonies to recover the ligated plasmids-C1, or C2 inserts </h4></a></br> |
| − | <a href="# | + | <a href="#exp15"><h4>62. Ligation of pET43.1a(+) with C1 and C2</h4></a></br> |
| − | <a href="# | + | <a href="#exp16"><h4>63. Digestion of insert B2</h4></a></br> |
| − | <a href="# | + | <a href="#exp17"><h4>64. ligation of insert B2 in pET43.1a(+)</h4></a></br> |
</p> | </p> | ||
| − | <p><h3><B>July 7, 2016: </B></h3></p> | + | <p><h3><B>July 7, 2016: </B></h3></p> |
<p> | <p> | ||
| − | <a href="# | + | <a href="#exp18"><h4> 65. Check of the petri dish done on the July 6, 2016 </h4></a></br> |
| − | <a href="# | + | <a href="#exp19"><h4> 66. Extraction of DNA of colonies from 06/07/2016 </h4></a></br> |
| − | <a href="# | + | <a href="#exp20"><h4> 67. Dosage of extracted DNA </h4></a></br> |
| − | + | <a href="#exp21"><h4> 69. Digestion of extracted DNA </h4></a></br> | |
| − | <a href="# | + | <a href="#exp22"><h4> 70. Electrophoresis of pET43.1a(+) </h4></a></br> |
| − | <a href="# | + | <a href="#exp23"><h4> 71. Digestion of the recombinating plasmid </h4></a></br> |
| − | <a href="# | + | <a href="#exp24"><h4> 72. Electrophoresis of our results</h4></a></br> |
| − | <a href="# | + | |
</p> | </p> | ||
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<p><h3><B>July 8, 2016: </B></h3></p> | <p><h3><B>July 8, 2016: </B></h3></p> | ||
<p> | <p> | ||
| − | <a href="# | + | <a href="#exp25"><h4> 73. Transformation of bacteria BL21DE3 </h4></a></br> |
</p> | </p> | ||
| Line 279: | Line 288: | ||
<div class="lightbox" id="exp1"> | <div class="lightbox" id="exp1"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We do again the previous experiment to understand why transformation in our bacteria does not work.</br></br> | + | <h6><U> Aim:</U></h6> We do again the previous experiment to understand why transformation in our bacteria does not work.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a(+)a plamid (obtained with Midiprep done on June 8, 2016)</br> | |
| − | + | • enzyme restriction (XbaI / HindIII)</br> | |
| − | + | • Buffer Cutsmart 10X (NEB)</br> | |
| − | + | • H<sub>2</sub>O</br> | |
| − | + | • P10 pipet and P20 pipet <br /> | |
| + | • 1.5 ml eppendorf <br /> | ||
| + | • 37°C and 65°C water bath </br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. For our manipulation, we used XbaI and HindIII enzymes and we used Cutsmart 10 X buffer because it is the most appropriate buffer.</br> | 1. For our manipulation, we used XbaI and HindIII enzymes and we used Cutsmart 10 X buffer because it is the most appropriate buffer.</br> | ||
| Line 298: | Line 309: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 38</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th></th> | <th></th> | ||
| − | <th>pET43.1a(+) à 130 ng/ | + | <th>pET43.1a(+) à 130 ng/µ (3 µg)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA ( | + | <td align = "center"; valign = "center">><strong><p>DNA (µl)</p></strong></td> |
| − | <td>19</td> | + | <td align = "center"; valign = "center">19</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>XbaI ( | + | <td align = "center"; valign = "center"><strong>XbaI (µl) </strong></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Hind III ( | + | <td align = "center"; valign = "center"><strong>Hind III (µl)</strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> | + | <td align = "center"; valign = "center"><strong>H<sub>20</sub< (µl)</strong></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>CutSmart 10X ( | + | <td align = "center"; valign = "center"><strong>CutSmart 10X (µl)</strong></td> |
| − | <td>24.5</td> | + | <td align = "center"; valign = "center">24.5</td> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>TOTAL ( | + | <td align = "center"; valign = "center"><strong>TOTAL (µl) </strong></td> |
| − | <td>51.5</td> | + | <td align = "center"; valign = "center">51.5</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | ||
</br></br></br> | </br></br></br> | ||
</p> | </p> | ||
| Line 343: | Line 354: | ||
<div class="lightbox" id="exp2"> | <div class="lightbox" id="exp2"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 39</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th></th> | <th></th> | ||
<th> = 260 nm</th> | <th> = 260 nm</th> | ||
| − | <th>pET43.1a(+) | + | <th>pET43.1a(+) XbaI/HindIII</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>N°1</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>N°1</p></strong></td> |
| − | <td>A260/280</td> | + | <td align = "center"; valign = "center">A260/280</td> |
| − | <td>1.8</td> | + | <td align = "center"; valign = "center">1.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>N°1</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>N°1</p></strong></td> |
| − | <td>Cfinal</td> | + | <td align = "center"; valign = "center">Cfinal</td> |
| − | <td>260 ng/ | + | <td align = "center"; valign = "center">260 ng/µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>N°2</strong></td> | + | <td align = "center"; valign = "center"><strong>N°2</strong></td> |
| − | <td>A260/A280</td> | + | <td align = "center"; valign = "center">A260/A280</td> |
| − | <td>1.4</td> | + | <td align = "center"; valign = "center">1.4</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>N°2</strong></td> | + | <td align = "center"; valign = "center"><strong>N°2</strong></td> |
| − | <td>Cfinal</td> | + | <td align = "center"; valign = "center">Cfinal</td> |
| − | <td>400 ng/ | + | <td align = "center"; valign = "center">400 ng/µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | ||
</br></br></br> | </br></br></br> | ||
</p> | </p> | ||
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<div class="lightbox" id="exp3"> | <div class="lightbox" id="exp3"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | < | + | <h6><U> Aim:</U></h6> Check if digestion was successfully done |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</br></br> | </br></br> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a(+) plasmid (obtained with Midiprep on june 8, 2016)</br> | |
| − | + | • pET43.1a(+) plasmid digested by XbaI/HindIII</br> | |
| − | + | • gel 0.7% agarose</br> | |
| − | + | • TAE 0.5X buffer</br> | |
| − | + | • Electrophoresis generator (at 50 V and after at 90 V)</br> | |
| − | + | • DNA ladder (Thermoscientific gene ruler 1kb)</br> | |
| − | + | • P10 and P20 pipet <br /> | |
| + | • 1.5 eppendorf </br> | ||
| + | • Electrophoresis BIORAD Mini-Sub Cell GT<br /><br /> | ||
| − | <U>Method:</U></br> | + | <h6><U>Method:</U></h6></br> |
1- Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts) </br> | 1- Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts) </br> | ||
2- Fill electrophoresis chamber with TAE 1X</br> | 2- Fill electrophoresis chamber with TAE 1X</br> | ||
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<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 40</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Marker weight</td> | + | <td align = "center"; valign = "center">Marker weight</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+) X/H</td> | + | <td align = "center"; valign = "center">pET43.1a(+) X/H</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+) UNCUT</td> | + | <td align = "center"; valign = "center">pET43.1a(+) UNCUT</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>A<sub>DNA (µl)</sub></strong></td> | + | <td align = "center"; valign = "center"><strong>A<sub>DNA (µl)</sub></strong></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong> | + | <td align = "center"; valign = "center"><strong>H<sub>2</sub>0 (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></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Load buffer 6X</strong></td> | + | <td align = "center"; valign = "center"><strong>Load buffer 6X</strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>2</td> | + | <td align = "center"; valign = "center">2</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
| − | + | </br></br> | |
| − | + | ||
| − | + | ||
| − | + | ||
1- Put the voltmeter at 100 V for one hour</br> | 1- Put the voltmeter at 100 V for one hour</br> | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6></br> |
| − | + | <center><img src = "https://static.igem.org/mediawiki/2016/f/ff/Week_5_Experience_50_Figure_7.jpg"; alt = "Digestion gel of pET43.1(a+)"/> | |
| − | + | <i><p><U>Figure 7 :</U> Digestion gel of pET43.1(a+)</p></i></center> | |
| − | + | We saw that in the line of the plasmid cut by XbaI and HindIII, we have two bands which don’t correspond to the uncut plasmid band. Moreover, The band of the plasmid cut by XbaI and HindIII have the appropriate weight. So we can cuclude that the digestion has worked. We can get back our plasmid cut with extraction gel.<br /> | |
| − | We saw that in the line of the plasmid cut by XbaI and HindIII, we have two bands which don’t correspond to the uncut plasmid band. Moreover, The band of the plasmid cut by XbaI and HindIII have the appropriate weight. So we can cuclude that the digestion has worked. We can get back our plasmid cut with extraction gel. | + | <br /><br /><br /> |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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</div> | </div> | ||
| − | <div class="lightbox" id=" | + | |
| + | |||
| + | <div class="lightbox" id="exp4"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>To recover the pET43.1a(+) which has been digested, and to purify out the band from the gel.</br></br> | + | <h6><U> Aim:</U></h6>To recover the pET43.1a(+) which has been digested, and to purify out the band from the gel.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • Results of electrophoresis (done previously)</br> | |
| − | + | • Gel extraction kit from Qiagen</br> | |
| − | + | • P10 and P20 pipet, 1.5 ml Eppendorf</br> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
To extract DNA we use the Gel Extraction Kit from Qiagen and we follow the different steps detailed in the kit.</br></br> | To extract DNA we use the Gel Extraction Kit from Qiagen and we follow the different steps detailed in the kit.</br></br> | ||
| − | + | • Eppendorf mass : 1.4043 g</br> | |
| − | + | • Eppendorf + Gel mass : 1.0817 g</br> | |
| − | + | • Gel mass : 1.4043 – 1.0817 = 322.6 mg</br></br> | |
We must pour 3 volums of QG buffer, it means 967.8 µl.</br> | We must pour 3 volums of QG buffer, it means 967.8 µl.</br> | ||
| − | We added 322 | + | We added 322 µl of isopropanol and we split in two equals volums our experiment</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 41</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 516: | Line 519: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</p></strong></td> |
| − | <td>322.6</td> | + | <td align = "center"; valign = "center">322.6</td> |
| − | <td>1.073</td> | + | <td align = "center"; valign = "center">1.073</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | < | + | <br /><br /> |
| − | </br></br></br> </p> | + | </br></br></br> |
| + | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp5"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>After the electrophoresis, we saw that the digestion of pET43.1a(+) was done succesfully. Now we have to dephosphorylate it to avoid self-ligation. </br></br> | + | <h6><U> Aim:</U></h6> After the electrophoresis, we saw that the digestion of pET43.1a(+) was done succesfully. Now we have to dephosphorylate it to avoid self-ligation. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a(+) plasmid digested by XbaI/HindIII</br> | |
| − | + | • Dephosphorylation rSAP enzyme</br> | |
| − | + | • P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)</br> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. For volumes, refer on the next table : </br> | 1. For volumes, refer on the next table : </br> | ||
| − | We dephosphorylated 7.5*120 ng/ | + | We dephosphorylated 7.5*120 ng/µl, so 900 ng of pET43.1a(+)</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 42</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 559: | Line 564: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td>7.5</td> | + | <td align = "center"; valign = "center">7.5</td> |
| − | <td>2.58</td> | + | <td align = "center"; valign = "center">2.58</td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
| − | <td>43.92</td> | + | <td align = "center"; valign = "center">43.92</td> |
| − | <td>60</td> | + | <td align = "center"; valign = "center">60</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 574: | Line 579: | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp6"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>To save time, we do another dephosphorylation of pET43.1 digested by enzymes to ligate it to the insert C2 (digest by XbaI/HindIII on June,28 2016). </br></br> | + | <h6><U> Aim:</U></h6>To save time, we do another dephosphorylation of pET43.1 digested by enzymes to ligate it to the insert C2 (digest by XbaI/HindIII on June,28 2016). </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a plasmid digested by XbaI/HindIII</br> | |
| − | + | • Dephosphorylation rSAP enzyme</br> | |
| − | + | • P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)</br><br /> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. For volumes, refer on the next table: | 1. For volumes, refer on the next table: | ||
| − | 2. We dephosphorylate 7.5*120 ng/ | + | 2. We dephosphorylate 7.5*120 ng/µl, so 900 ng of pET43.1</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 43</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 605: | Line 611: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td>7.5</td> | + | <td align = "center"; valign = "center">7.5</td> |
| − | <td>2.58</td> | + | <td align = "center"; valign = "center">2.58</td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
| − | <td>43.92</td> | + | <td align = "center"; valign = "center">43.92</td> |
| − | <td>60</td> | + | <td align = "center"; valign = "center">60</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | <br /><br /> | |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 619: | Line 626: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp7"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We want to obtain a second expression vector, this time with pET43.1 and C2. We do the same experiment as previously performed for C1.</br></br> | + | <h6><U> Aim:</U></h6> We want to obtain a second expression vector, this time with pET43.1 and C2. We do the same experiment as previously performed for C1.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylated</br> | |
| − | + | • C2 insert cut by XbaI/HindIII (done on the June,28 2016)</br> | |
| − | + | • T4 Ligase and Buffer 10X</br> | |
| − | + | • P10 and P200 pipet, 1.5 eppendorf, waterbath (37 °C and 65 °C)</br><br /> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. For volumes, refer to the next table:</br> | 1. For volumes, refer to the next table:</br> | ||
| − | C2 = 9.2 ng/ | + | C2 = 9.2 ng/µl</br> |
We used 60 µl of pET43.1a(+) concentrated at 15 ng/µl (total 900 ng)</br></br> | We used 60 µl of pET43.1a(+) concentrated at 15 ng/µl (total 900 ng)</br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 43</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 650: | Line 658: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+) (50 ng)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+) (50 ng)</p></strong></td> |
| − | <td>6.7</td> | + | <td align = "center"; valign = "center">6.7</td> |
| − | <td>6.7</td> | + | <td align = "center"; valign = "center">6.7</td> |
| − | <td>6.7</td> | + | <td align = "center"; valign = "center">6.7</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Insert (C2) ( | + | <td align = "center"; valign = "center"><strong>Insert (C2) (µl)</strong></td> |
| − | <td>1.74</td> | + | <td align = "center"; valign = "center">1.74</td> |
| − | <td>5.22</td> | + | <td align = "center"; valign = "center">5.22</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>T4 Ligase ( | + | <td align = "center"; valign = "center"><strong><p>T4 Ligase (µl)</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer 10X (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X (µl)</p></strong></td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
| − | <td>1.5</td> | + | <td align = "center"; valign = "center">1.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>H20 (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>H20 (µl)</p></strong></td> |
| − | <td>4.06</td> | + | <td align = "center"; valign = "center">4.06</td> |
| − | <td>0.58</td> | + | <td align = "center"; valign = "center">0.58</td> |
| − | <td>5.8</td> | + | <td align = "center"; valign = "center">5.8</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>TOTAL (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>TOTAL (µl)</p></strong></td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
| − | <td>15</td> | + | <td align = "center"; valign = "center">15</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
2. Incubate 10 min at room temperature, and put it at 65 °C during 10 min to inactivate the ligase. To keep it and use it after, we put it at -20 °C</br></br> | 2. Incubate 10 min at room temperature, and put it at 65 °C during 10 min to inactivate the ligase. To keep it and use it after, we put it at -20 °C</br></br> | ||
| − | + | <br /><br /><br /> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 697: | Line 702: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp8"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>At this stage, we want to obtain a recombinant vector (pET43.1a(+) + C2)</br></br> | + | <h6><U> Aim:</U></h6>At this stage, we want to obtain a recombinant vector (pET43.1a(+) + C2)</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylate (done previously)</br> | |
| − | + | • C2 insert cut by XbaI/HindIII (done on the june,28 2016)</br> | |
| − | + | • T4 Ligase and Buffer 10X</br> | |
| − | + | • P10 and P200 pipet, 1.5 eppendorf, warm bath (37 °C and 65 °C)</br><br /> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. For volums, refer to the next table : | + | 1. For volums, refer to the next table :</br></br> |
| − | C2 = 11.4 ng/ | + | C2 = 11.4 ng/µl </br> |
| − | pET43.1 = 50 ng/ | + | pET43.1 = 50 ng/µl (900 ng in 60 µl) </br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 44</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 729: | Line 735: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>pET43.1a(+) (50mg) (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>pET43.1a(+) (50mg) (µl)</p></strong></td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
| − | <td>20</td> | + | <td align = "center"; valign = "center">20</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Insert C2 ( | + | <td align = "center"; valign = "center"><strong><p>Insert C2 (µl)</p></strong></td> |
| − | <td>1.4</td> | + | <td align = "center"; valign = "center">1.4</td> |
<td>4.2</td> | <td>4.2</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>T4 ligase ( | + | <td align = "center"; valign = "center"><strong><p>T4 ligase (µl) </p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer 10X ( | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X (µl) </p></strong></td> |
| − | <td>2.5</td> | + | <td align = "center"; valign = "center">2.5</td> |
| − | <td>2.5</td> | + | <td align = "center"; valign = "center">2.5</td> |
| − | <td>2.5</td> | + | <td align = "center"; valign = "center">2.5</td> |
</tr> | </tr> | ||
<tr> <tr> | <tr> <tr> | ||
| − | <td><strong><p>H20 ( | + | <td align = "center"; valign = "center"><strong><p>H20 (µl) </p></strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</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><p>TOTAL ( | + | <td align = "center"; valign = "center"><strong><p>TOTAL (µl) </p></strong></td> |
| − | <td>24.9</td> | + | <td align = "center"; valign = "center">24.9</td> |
| − | <td>27.7</td> | + | <td align = "center"; valign = "center">27.7</td> |
| − | <td>22.5</td> | + | <td align = "center"; valign = "center">22.5</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
Incubate 10 min at room temperature, and put it at 37 °C during 10 min. To keep it and use it after, we put it at -20 °C. </br> | Incubate 10 min at room temperature, and put it at 37 °C during 10 min. To keep it and use it after, we put it at -20 °C. </br> | ||
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 776: | Line 782: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp9"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Check if ligation of pET43.1+C1 and pET43.1+C2 were successfully done | + | <h6><U> Aim:</U></h6> Check if ligation of pET43.1+C1 and pET43.1+C2 were successfully done.</br></br> |
| − | </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> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>Materials:</U></h6> |
| − | <U>Materials:</U></ | + | • pET43.1a plamid </br> |
| − | + | • pET43.1a plamid cutted by HindIII/XbaI</br> | |
| − | + | • C1 and C2 cutted by HindIII/XbaI</br> | |
| − | + | • agarose gel 0.7%</br> | |
| − | + | • TAE 0.5x buffer</br> | |
| − | + | • Electrophoresis generator at 130 V</br> | |
| − | + | • DNA ladder (Thermoscientific gene ruler 1 kb)</br> | |
| − | + | • Electrophoresis generator (at 50 V and after at 90 V)</br> | |
| − | + | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts) </br> | 1. Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts) </br> | ||
2. Fill electrophoresis chamber with TAE 0.5X</br> | 2. Fill electrophoresis chamber with TAE 0.5X</br> | ||
| Line 801: | Line 806: | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 45</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 817: | Line 823: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Marker weight</td> | + | <td align = "center"; valign = "center">Marker weight</td> |
| − | <td>pET43.1a(+) uncul</td> | + | <td align = "center"; valign = "center">pET43.1a(+) uncul</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C1 (1:1)</td> | + | <td align = "center"; valign = "center">C1 (1:1)</td> |
| − | <td>C1 (1:3)</td> | + | <td align = "center"; valign = "center">C1 (1:3)</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (1:1)</td> | + | <td align = "center"; valign = "center">C2 (1:1)</td> |
| − | <td>C2 (1:3)</td> | + | <td align = "center"; valign = "center">C2 (1:3)</td> |
| − | <td>pET43.1a(+) only</td> | + | <td align = "center"; valign = "center">pET43.1a(+) only</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>7</td> | + | <td align = "center"; valign = "center">7</td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
<td></td> | <td></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>0</td> | + | <td align = "center"; valign = "center">0</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></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>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | + | ||
Let 1h at 50V and put it at 100 V.</br></br> | Let 1h at 50V and put it at 100 V.</br></br> | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6></br> |
| − | The electrophoresis shows us that DNA wasn’t ligated, so we added 1 µl more of T4 ligase and 1 | + | The electrophoresis shows us that DNA wasn’t ligated, so we added 1 µl more of T4 ligase and 1 µl of buffer in each tube and we let ligate during 1 hour. </br></br> |
| − | After one hour, we did another electrophoresis.</br></br> | + | After one hour, we did another electrophoresis.</br> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 880: | Line 885: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp10"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Check if ligation of pET43.1+C1 and pET43.1+C2 was successfully done.</br></br> | + | <h6><U> Aim:</U></h6>Check if ligation of pET43.1+C1 and pET43.1+C2 was successfully done.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • pET43.1a plamid </br> | |
| − | + | • pET43.1a plamid cutted by HindIII/XbaI</br> | |
| − | + | • C1 and C2 cut by HindIII/XbaI</br> | |
| − | + | • agarose gel 0.7%</br> | |
| − | + | • TAE 0.5x buffer</br> | |
| − | + | • Electrophoresis generator at 130 V</br> | |
| − | + | • DNA ladder (Thermoscientific gene ruler 1 kb)</br> | |
| − | + | • P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D, </br></br> | |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. -Fill the electrophoresis chamber with TAE 0.5X buffer</br> | 1. -Fill the electrophoresis chamber with TAE 0.5X buffer</br> | ||
2. Following this deposit table :</br></br> | 2. Following this deposit table :</br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 46</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 917: | Line 923: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Marker weight</td> | + | <td align = "center"; valign = "center">Marker weight</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+) uncul</td> | + | <td align = "center"; valign = "center">pET43.1a(+) uncul</td> |
| − | <td>pET43.1a(+) only</td> | + | <td align = "center"; valign = "center">pET43.1a(+) only</td> |
| − | <td>C1 (1:1)</td> | + | <td align = "center"; valign = "center">C1 (1:1)</td> |
| − | <td>C1 (1:3)</td> | + | <td align = "center"; valign = "center">C1 (1:3)</td> |
| − | <td>C2 (1:1)</td> | + | <td align = "center"; valign = "center">C2 (1:1)</td> |
| − | <td>C2 (1:3)</td> | + | <td align = "center"; valign = "center">C2 (1:3)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
| − | <td>5</td> | + | <td align = "center"; valign = "center">5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></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>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</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><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | + | <br /><br /> | |
| − | + | </br></br><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 972: | Line 978: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp11"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6> |
MpET43.1 = 5 300 bp * 660 g.mol-1.bp-1</br> | MpET43.1 = 5 300 bp * 660 g.mol-1.bp-1</br> | ||
MpET43.1 = 3.5*10-6 g/mol</br> | MpET43.1 = 3.5*10-6 g/mol</br> | ||
| Line 987: | Line 993: | ||
- We have 0.12 µmol/L in 20 µl, and we must have 10 pg to have a good yield in colonies. </br> | - We have 0.12 µmol/L in 20 µl, and we must have 10 pg to have a good yield in colonies. </br> | ||
- Finally, we have 0.12 * 20*10-6 = 2.4*10-6 µmol of plasmid and 8.16 pg of plasmid. </br> | - Finally, we have 0.12 * 20*10-6 = 2.4*10-6 µmol of plasmid and 8.16 pg of plasmid. </br> | ||
| − | </br> | + | </br><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 994: | Line 1,000: | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp12"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> To make stock cells containing the plasmids, we need to enter the plasmid with C1 and C2 insert into the bacteria DH5 competent cells.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | <U>Method:</U></ | + | • Competent cells<br /> |
| + | • SOC media <br /> | ||
| + | •42°C waterbath<br /> | ||
| + | •LB/carbenicillin 50 µg/ml.</br></br> | ||
| + | <h6><U>Method:</U></h6> | ||
1- In five 1.5 ml eppendorf tubes, we put 40 µl of DH5 competent cells and we add 5µl of : </br> | 1- In five 1.5 ml eppendorf tubes, we put 40 µl of DH5 competent cells and we add 5µl of : </br> | ||
(vector:Insert) ratio</br> | (vector:Insert) ratio</br> | ||
| Line 1,012: | Line 1,022: | ||
(5) C2 (1:3) </br></br> | (5) C2 (1:3) </br></br> | ||
| − | 2- Place the tubes on ice during 30 min ( | + | 2- Place the tubes on ice during 30 min (6:30<sub>PM</sub>).</br> |
| − | 3- Put them at 42 °C during 40 sec</br> | + | 3- Put them at 42 °C during 40 sec.</br> |
| − | + | 4- Put them on ice during 2 min 30 sec.</br> | |
| − | + | 5- Add 200 µl of SOC in each tubes and place them in a shaking incubator for 1 h at 37°C and at 225 RPM.</br> | |
| − | + | 6- We spread our mix (250 µl) on petri dishes made of LB and carbenicillin. </br> | |
| − | + | 7- Put them at 37 °C for one night.</br> | |
| − | + | <br /></br></br> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,025: | Line 1,035: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp13"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
Only two colonies have grown on the plate C2 (1:3), all the other petri dishes are empty. </br> | Only two colonies have grown on the plate C2 (1:3), all the other petri dishes are empty. </br> | ||
| − | According to the electrophoresis gel, the ligation has worked for C1 (1:3) and C2 (1:1) but it do not correspond to our results. We decided to pool C2 (1:1) with C2 (1:3) and C1 (1:1) with C1 (1:3) because the gel shows the same efficiency of ligation, in order to have more DNA for transformation. | + | According to the electrophoresis gel, the ligation has worked for C1 (1:3) and C2 (1:1) but it do not correspond to our results. We decided to pool C2 (1:1) with C2 (1:3) and C1 (1:1) with C1 (1:3) because the gel shows the same efficiency of ligation, in order to have more DNA for transformation.</br></br> |
</p> | </p> | ||
| Line 1,039: | Line 1,049: | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp14"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We want to increase the number of bacteria to check if they have the right plasmid. As we have two colonies, therefore we placed them to grow in liquid media in two Falcons. </br></br> | + | <h6><U> Aim:</U></h6> We want to increase the number of bacteria to check if they have the right plasmid. As we have two colonies, therefore we placed them to grow in liquid media in two Falcons. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_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> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
In a 50 ml Falcon : </br> | In a 50 ml Falcon : </br> | ||
- Put 10 ml of LB and 5 µl of carbenicillin at 100 mg/ml. </br> | - Put 10 ml of LB and 5 µl of carbenicillin at 100 mg/ml. </br> | ||
| Line 1,053: | Line 1,063: | ||
- Put the Falcon in a shaking incubator at 37 °C for one day. </br></br> | - Put the Falcon in a shaking incubator at 37 °C for one day. </br></br> | ||
| − | + | </br></br> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,060: | Line 1,070: | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp15"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
The ligation does not work well, so we pooled our ligation products to increase the amount of available DNA. | The ligation does not work well, so we pooled our ligation products to increase the amount of available DNA. | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> At this stage, we want to obtain a ligated vector (pET43.1a(+) + C1/C2)</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
C(pET43.1) = 29.5 ng/µl</br> | C(pET43.1) = 29.5 ng/µl</br> | ||
C(C1) = 11.4 ng/µl</br> | C(C1) = 11.4 ng/µl</br> | ||
C(C2) = 9.2 ng/µl</br></br> | C(C2) = 9.2 ng/µl</br></br> | ||
| − | + | Add volumes of 1µl of T4 Ligase and 1 µl of Buffer 10X in the tubes C1(1 :1)+C1(1 :3) and C2(1 :1)+C2(1 :3).</br> | |
| − | Let the ligation proceed during 1h30 at RT and incubate it 10 min at 65 °C to inactivated the ligase.</br> | + | Let the ligation proceed during 1h30 at RT and incubate it 10 min at 65 °C to inactivated the ligase.</br></br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,083: | Line 1,093: | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp16"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>The | + | <h6><U> Aim:</U></h6>The previous experiment being still underway, we want to move ahead wier inserts. We want to digest our B2 insert to put it in the expression vector.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • eppendorf (0.5 ml)</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> | |
| − | + | • B2 insert</br> | |
| − | + | • Enzymes (HindIII and XbaI)</br> | |
| − | + | • H2O RNAse free</br> | |
• Buffer 10X</br></br> | • Buffer 10X</br></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Mixing all of reactants and let the digestion proceed during 1h30 at | + | 1. Mixing all of reactants and let the digestion proceed during 1h30 at 37°C </br> |
2. Follow this next table</br></br> | 2. Follow this next table</br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 47</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,110: | Line 1,121: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA (50ng/µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA (50ng/µl)</p></strong></td> |
| − | <td>20 µl</td> | + | <td align = "center"; valign = "center">20 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Vol Hind III</strong></td> | + | <td align = "center"; valign = "center"><strong>Vol Hind III</strong></td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>A<sub>Vol XbaI</sub></strong></td> | + | <td align = "center"; valign = "center"><strong>A<sub>Vol XbaI</sub></strong></td> |
| − | <td>0.5 µl</td> | + | <td align = "center"; valign = "center">0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Vol buffer 10X (CutSmart)</strong></td> | + | <td align = "center"; valign = "center"><strong>Vol buffer 10X (CutSmart)</strong></td> |
| − | <td>2.5 µl</td> | + | <td align = "center"; valign = "center">2.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>Vol TOTAL</strong></td> | + | <td align = "center"; valign = "center"><strong>Vol TOTAL</strong></td> |
| − | <td>25 µl</td> | + | <td align = "center"; valign = "center">25 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | Allow the digestion proceed for 1h and add an extra 0.5 µl of enzyme and leave the reaction going for 1h more at | + | 1. Allow the digestion proceed for 1h and add an extra 0.5 µl of enzyme and leave the reaction going for 1h more at 37°C.</br> |
| − | + | 2. Incubate it 10 min at 65 °C to inactivate the enzymes.</br> | |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,140: | Line 1,151: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp17"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Than we do the ligation of the insert B2 with pET43.1 and the transformation of all our products of ligation B2/C1 mix and empty plasmid as control. </br></br> | + | <h6><U> Aim:</U></h6>Than we do the ligation of the insert B2 with pET43.1 and the transformation of all our products of ligation B2/C1 mix and empty plasmid as control. </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></br><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,154: | Line 1,166: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp18"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U>Results:</U></ | + | <h6><U>Results:</U></h6></br> |
B2 (1 : 1) Nothing has grown</br> | B2 (1 : 1) Nothing has grown</br> | ||
B2 (1 : 3) Nothing has grown</br> | B2 (1 : 3) Nothing has grown</br> | ||
C2 (1 : 0) Nothing has grown</br> | C2 (1 : 0) Nothing has grown</br> | ||
C1 mix Nothing has grown</br> | C1 mix Nothing has grown</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,170: | Line 1,182: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp19"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>To get the plasmid back and verify that it contains inserts. | + | <h6><U> Aim:</U></h6> To get the plasmid back and verify that it contains inserts.<br /> |
We have two colonies, so for each colony, we started two cultures in Falcon tubes. One being a primary culture and the other a backup, in case the culture doesn't grow. | We have two colonies, so for each colony, we started two cultures in Falcon tubes. One being a primary culture and the other a backup, in case the culture doesn't grow. | ||
</br></br> | </br></br> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_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> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
They are named :</br> | They are named :</br> | ||
C2 (1 :3) 1.1</br> | C2 (1 :3) 1.1</br> | ||
| Line 1,187: | Line 1,199: | ||
C2 (1 :3) 2.2</br></br> | C2 (1 :3) 2.2</br></br> | ||
| − | NB : | + | <i>NB :The naming convention here is first number is the number of colony, and the second number correspond to primary 1 ou secondary 2 cultures.</i></br> |
| − | + | <br /><br /><br /> | |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,196: | Line 1,207: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | |
| + | <div class="lightbox" id="exp20"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> | + | <h6><U> Aim:</U></h6>We want to check if our bacteria have produced enought ligated plasmid. </br></br> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <h6><U>What we did in the lab:</U></h6></br> | |
| − | + | </br></br> | |
| − | + | <h6><U>Method:</U></h6> | |
| − | + | ||
| − | + | ||
| − | < | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | <U>Method:</U></ | + | |
Spectro : Ultrospec 3100 pro-Amersham Bioscience</br> | Spectro : Ultrospec 3100 pro-Amersham Bioscience</br> | ||
Vol DNA = 2 l</br> | Vol DNA = 2 l</br> | ||
| Line 1,226: | Line 1,227: | ||
- use 2 µl DNA in 998 µl of TE for the dilution</br> | - use 2 µl DNA in 998 µl of TE for the dilution</br> | ||
Analysis to = 260 nm</br> | Analysis to = 260 nm</br> | ||
| − | Blank on TE1X </br> | + | Blank on TE1X </br></br> |
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 48</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,241: | Line 1,243: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinale></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinale></p></strong></td> |
| − | <td>110 ng/µl</td> | + | <td align = "center"; valign = "center">110 ng/µl</td> |
| − | <td>290 ng/µl</td> | + | <td align = "center"; valign = "center">290 ng/µl</td> |
| − | <td>110 ng/µl</td> | + | <td align = "center"; valign = "center">110 ng/µl</td> |
| − | <td>70 ng/µl</td> | + | <td align = "center"; valign = "center">70 ng/µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Cfinale2></p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Cfinale2></p></strong></td> |
| − | <td>5 500 ng/µl</td> | + | <td align = "center"; valign = "center">5 500 ng/µl</td> |
| − | <td>14 500 ng/µl</td> | + | <td align = "center"; valign = "center">14 500 ng/µl</td> |
| − | <td>5 500 ng/µl</td> | + | <td align = "center"; valign = "center">5 500 ng/µl</td> |
| − | <td>3 500 ng/µl</td> | + | <td align = "center"; valign = "center">3 500 ng/µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | < | + | </table><br /><br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,261: | Line 1,264: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp21"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> We want to check if the plasmid has an insert.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> |
| − | + | <h6><U>Method:</U></h6> | |
| − | <U>Method:</U></ | + | |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 49</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,283: | Line 1,287: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (1 µg)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (1 µg)</p></strong></td> |
| − | <td>9.1 µl</td> | + | <td align = "center"; valign = "center">9.1 µl</td> |
| − | <td>3.4 µl</td> | + | <td align = "center"; valign = "center">3.4 µl</td> |
| − | <td>9.1 µl</td> | + | <td align = "center"; valign = "center">9.1 µl</td> |
| − | <td>14.3µl</td> | + | <td align = "center"; valign = "center">14.3µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>XbaI</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>XbaI</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>1 µl</td> | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>HindIII</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>HindIII</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>1 µl</td> | + | <td align = "center"; valign = "center">1 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>15.9 µl</td> | + | <td align = "center"; valign = "center">15.9 µl</td> |
| − | <td>21.6 µl</td> | + | <td align = "center"; valign = "center">21.6 µl</td> |
| − | <td>15.9 µl</td> | + | <td align = "center"; valign = "center">15.9 µl</td> |
| − | <td>10.7 µl</td> | + | <td align = "center"; valign = "center">10.7 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer 10X</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X</p></strong></td> |
| − | <td>3 µl</td> | + | <td align = "center"; valign = "center">3 µl</td> |
| − | <td>3 µl</td> | + | <td align = "center"; valign = "center">3 µl</td> |
| − | <td>3 µl</td> | + | <td align = "center"; valign = "center">3 µl</td> |
| − | <td>3 µl</td> | + | <td align = "center"; valign = "center">3 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer 10X</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X</p></strong></td> |
| − | <td>30 µl</td> | + | <td align = "center"; valign = "center">30 µl</td> |
| − | <td>30 µl</td> | + | <td align = "center"; valign = "center">30 µl</td> |
| − | <td>30 µl</td> | + | <td align = "center"; valign = "center">30 µl</td> |
| − | <td>30 µl</td> | + | <td align = "center"; valign = "center">30 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | + | </table></br></br> | |
| − | 1. Add all reagents in a 1.5 ml eppendorf </br> | + | 1. Add all reagents in a 1.5 ml eppendorf. </br> |
| − | 2. Let the digestion proceed during 1h30 at | + | 2. Let the digestion proceed during 1h30 at 37°C and incubate 10 min at 65°C.</br> |
| − | 3. For the reagent volumes, refer to the table</br> | + | 3. For the reagent volumes, refer to the table.</br> |
| + | </br><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,334: | Line 1,339: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp22"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We want to check if the plasmid has an insert. </br></br> | + | <h6><U> Aim:</U></h6> We want to check if the plasmid has an insert. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
<U>Method:</U></br> | <U>Method:</U></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 50</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,362: | Line 1,368: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Mark weight</td> | + | <td align = "center"; valign = "center">Mark weight</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1a(+) X/H</td> | + | <td align = "center"; valign = "center">pET43.1a(+) X/H</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (1:1)</td> | + | <td align = "center"; valign = "center">C2 (1:1)</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (1:2)</td> | + | <td align = "center"; valign = "center">C2 (1:2)</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (2:1)</td> | + | <td align = "center"; valign = "center">C2 (2:1)</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (2:2)</td> | + | <td align = "center"; valign = "center">C2 (2:2)</td> |
| − | <td>pET43.1a(+) uncut</td> | + | <td align = "center"; valign = "center">pET43.1a(+) uncut</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>6</td> | + | <td align = "center"; valign = "center">6</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
| − | <td>30</td> | + | <td align = "center"; valign = "center">30</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | + | </table></br></br> | |
| − | + | ||
| − | + | <h6><U>Results:</U></h6></br> | |
After the elctrophoresis, we notice that the recombinating plasmid seems to contain two inserts. Indeed, the band corresponds to a size between 1 00 and 1 500 bp.</br> | After the elctrophoresis, we notice that the recombinating plasmid seems to contain two inserts. Indeed, the band corresponds to a size between 1 00 and 1 500 bp.</br> | ||
We decided to digest our double insert with XbaI and SpeI to split it. </br> | We decided to digest our double insert with XbaI and SpeI to split it. </br> | ||
We also digested a pET43.1with XbaI and SpeI to fit it. </br> | We also digested a pET43.1with XbaI and SpeI to fit it. </br> | ||
| + | <br/><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,403: | Line 1,410: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp23"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We observed that two inserts were present in our gels, we therefore want to verify that the second heavier one is not as a result illegitimate ligation of XbaI/HindIII. There is a SpeI site in the neighboring sequences. Therefore, we want to split the insert to have just one insert.</br></br> | + | <h6><U> Aim:</U><:</h6>We observed that two inserts were present in our gels, we therefore want to verify that the second heavier one is not as a result illegitimate ligation of XbaI/HindIII. There is a SpeI site in the neighboring sequences. Therefore, we want to split the insert to have just one insert.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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. For volumes, refer to the next table :</br> | 1. For volumes, refer to the next table :</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 51</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,426: | Line 1,434: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol DNA (3 µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol DNA (3 µl)</p></strong></td> |
| − | <td>27.3 µl</td> | + | <td align = "center"; valign = "center">27.3 µl</td> |
| − | <td>10.3 µl</td> | + | <td align = "center"; valign = "center">10.3 µl</td> |
| − | <td>2.5 µl</td> | + | <td align = "center"; valign = "center">2.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol SepI (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol SepI (µl)</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol XbaI (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol XbaI (µl)</p></strong></td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
| − | <td>1</td> | + | <td align = "center"; valign = "center">1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>H20 10X (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>H20 10X (µl)</p></strong></td> |
| − | <td>6.7</td> | + | <td align = "center"; valign = "center">6.7</td> |
| − | <td>23.7</td> | + | <td align = "center"; valign = "center">23.7</td> |
| − | <td>31.5</td> | + | <td align = "center"; valign = "center">31.5</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Vol Buffer CutSmart 10X (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol Buffer CutSmart 10X (µl)</p></strong></td> |
| − | <td>4</td> | + | <td align = "center"; valign = "center">4</td> |
| − | <td>4</td> | + | <td align = "center"; valign = "center">4</td> |
| − | <td>4</td> | + | <td align = "center"; valign = "center">4</td> |
</tr> | </tr> | ||
| − | <td><strong><p>Vol TOTAL (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Vol TOTAL (µl)</p></strong></td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | + | </table></br></br> | |
| − | 2. After 1H of digestion, we added 1 µl of each enzyme and let digest 30 more minutes. | + | 2. After 1H of digestion, we added 1 µl of each enzyme and let digest 30 more minutes.</br> |
| + | </br><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,469: | Line 1,478: | ||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | <div class="lightbox" id="exp24"> |
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>We want to check if we succeeded in splitting the twinned insert.</br></br> | + | <h6><U> Aim:</U></h6>We want to check if we succeeded in splitting the twinned insert.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
1. To deposit volums, refer to this table :</br> | 1. To deposit volums, refer to this table :</br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 51</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,494: | Line 1,504: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Name</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Name</p></strong></td> |
| − | <td>Mark weight</td> | + | <td align = "center"; valign = "center">Mark weight</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>pET43.1 X/S</td> | + | <td align = "center"; valign = "center">pET43.1 X/S</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>C2 (1:1)</td> | + | <td align = "center"; valign = "center">C2 (1:1)</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
<td>C2 (1:2)</td> | <td>C2 (1:2)</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (µl)</p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>H20 (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>H20 (µl)</p></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></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>0</td> | + | <td align = "center"; valign = "center">0</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>40</td> | + | <td align = "center"; valign = "center">40</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>H20 (µl)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>H20 (µl)</p></strong></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td>10</td> | + | <td align = "center"; valign = "center">10</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | + | </table></br></br> | |
| − | <U>Results:</U>The digestion does not work, we did not succeed in splitting the twinned insert so we decided to keep it and to express the protein with the double insert, hoping that the stop codon at the end of one of the inserts will be ennough. We will also sequence the plasmid to verify the orientation, and validity of our twinned insert hypothesis.</br></br> | + | <h6><U>Results:</U></h6> The digestion does not work, we did not succeed in splitting the twinned insert so we decided to keep it and to express the protein with the double insert, hoping that the stop codon at the end of one of the inserts will be ennough. We will also sequence the plasmid to verify the orientation, and validity of our twinned insert hypothesis.</br> |
| + | </br><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
| − | |||
</div> | </div> | ||
| − | <div class="lightbox" id=" | + | |
| + | <div class="lightbox" id="exp27"> | ||
<figure> | <figure> | ||
| − | <a href="# | + | <a href="#" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>In this part we wantant to proceed with the expression of our fusion protein containing the R5-CBD-BPA. To do this we need a cell line having a T7 RNA polymerase. The cells we chose were BL21De3 pLys S. These contain the T7 phage producing the T7 RNA polymerase, and lysozyme, which will inhibit the T7 RNA polymrase and help control the expression better in case our protein is toxic to the cells, and secondly in case the cells escape our control they will not be able to survive in nature, as they will lyse over time.</br></br> | + | <h6><U> Aim:</U></h6> In this part we wantant to proceed with the expression of our fusion protein containing the R5-CBD-BPA. To do this we need a cell line having a T7 RNA polymerase. The cells we chose were BL21De3 pLys S. These contain the T7 phage producing the T7 RNA polymerase, and lysozyme, which will inhibit the T7 RNA polymrase and help control the expression better in case our protein is toxic to the cells, and secondly in case the cells escape our control they will not be able to survive in nature, as they will lyse over time.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | <U>Method:</U></ | + | • BL21De3 competent cells <br /> |
| − | (I) : 50 | + | • SOC media <br /> |
| − | (II) : 50 | + | • 42 °C waterbath.</br><br /> |
| − | (III) : 50 | + | <h6><U>Method:</U></h6> |
| − | (IV) : 50 | + | (I) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.1</br> |
| − | After heat shock at 42°C, the cells were allowed to recover by growing at 37°C for 40 minutes in 150 µl of SOC, then the 200 µl of each sample were spread on a petridish with carbenicillin and grown at 37 °C for one night.</br></br> | + | (II) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.2</br> |
| + | (III) : 50 µl of bacteria + 5 µl of pUC</br> | ||
| + | (IV) : 50 µl of bacteria + 5 µl of CT (plasmid given with the bacteria)</br></br></br> | ||
| + | After heat shock at 42°C, the cells were allowed to recover by growing at 37°C for 40 minutes in 150 µl of SOC, then the 200 µl of each sample were spread on a petridish with carbenicillin and grown at 37 °C for one night.</br> | ||
| + | </br><br /><br /> | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
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