Difference between revisions of "Team:Pasteur Paris/Microbiology week9"
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| + | <a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><h2><B>Microbiology Notebook</B></h2><a/> | ||
| + | </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="week9"> | <div id="week9"> | ||
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<p><h3><B> August 1, 2016:</B></h3></p> | <p><h3><B> August 1, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp1"><h4> Silification tests </h4></a><br/> | + | <a href="#exp1"><h4> 120. Silification tests </h4></a><br/> |
| − | <a href="#exp2"><h4> PCR of C1 v2 and C2 v2 with Takara enzyme </h4></a><br/> | + | <a href="#exp2"><h4> 121. PCR of C1 v2 and C2 v2 with Takara enzyme </h4></a><br/> |
| − | <a href="#exp3"><h4> Agarose gel </h4></a><br/> | + | <a href="#exp3"><h4> 122. Agarose gel </h4></a><br/> |
| − | <a href="#exp4"><h4> Electrophoresis of the PCR products (C1 v2 and C2 v2) </h4></a><br/> | + | <a href="#exp4"><h4> 123. Electrophoresis of the PCR products (C1 v2 and C2 v2) </h4></a><br/> |
| − | <a href= | + | <a href="#exp5"><h4> 124. PCR Clean up </h4></a><br/> |
</p> | </p> | ||
<p><h3><B> August 2, 2016:</B></h3></p> | <p><h3><B> August 2, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp6"><h4> PCR of A1, A2, B1, B2, D1, D2, E1 and C2 </h4></a><br/> | + | <a href="#exp6"><h4> 125. PCR of A1, A2, B1, B2, D1, D2, E1 and C2 </h4></a><br/> |
| − | <a href="#exp7"><h4> Analysis of PCR products from August 1 </h4></a><br/> | + | <a href="#exp7"><h4> 126. Analysis of PCR products from August 1 </h4></a><br/> |
| − | <a href="#exp8"><h4> PCR clean up </h4></a><br/> | + | <a href="#exp8"><h4> 127. PCR clean up </h4></a><br/> |
| − | <a href="#exp9"><h4> Ligation of PCR products with TOPO cloning </h4></a><br/> | + | <a href="#exp9"><h4> 128. Ligation of PCR products with TOPO cloning </h4></a><br/> |
| − | <a href= | + | <a href="#exp10"><h4> 129. Transformation in TOP10 competent cells </h4></a><br/> |
| − | <a href= | + | <a href="#exp11"><h4> 130. PCR of A1⁄A2 and D1⁄D2 </h4></a><br/> |
</p> | </p> | ||
| − | <p>< | + | <p><h3><B> August 3, 2016:</B></h3></p> |
<p> | <p> | ||
| − | <a href="#exp12"><h4> Analysis of PCR products from August 2, 2016 </h4></a><br/> | + | <a href="#exp12"><h4> 131. Analysis of PCR products from August 2, 2016 </h4></a><br/> |
| − | <a href="#exp13"><h4> Miniprep of cultures C1 v2 and C2 v2 from August 2 </h4></a><br/> | + | <a href="#exp13"><h4> 132. Miniprep of cultures C1 v2 and C2 v2 from August 2 </h4></a><br/> |
| − | <a href="#exp14"><h4> Digestion of C1 v2 and C2 v2 </h4></a><br/> | + | <a href="#exp14"><h4> 133. Digestion of C1 v2 and C2 v2 </h4></a><br/> |
| − | <a href="#exp15"><h4> Electrophoresis of C1 v2 and C2 v2 </h4></a><br/> | + | <a href="#exp15"><h4> 134. Electrophoresis of C1 v2 and C2 v2 </h4></a><br/> |
| − | <a href="#exp16"><h4> DNA extraction from the gel </h4></a><br/> | + | <a href="#exp16"><h4> 135. DNA extraction from the gel </h4></a><br/> |
| − | <a href="#exp17"><h4> Ligation of C1.2 | + | <a href="#exp17"><h4> 136. Ligation of C1.2⁄C1.5 and C2.1⁄C2.2 with pET43.1 (a+) and transformation with TOP10 competent cells </h4></a><br/> |
</p> | </p> | ||
| − | <p>< | + | <p><h3><B>August 4, 2016:</B></h3></p> |
<p> | <p> | ||
| − | <a href="#exp18"><h4> Miniprep of B1⁄B2 and E1⁄E2 </h4></a><br/> | + | <a href="#exp18"><h4> 137. Miniprep of B1⁄B2 and E1⁄E2 </h4></a><br/> |
| − | <a href="#exp19"><h4> Digestion of B1⁄B2, E1⁄E2 and pET43.1 with Hind III and Xba I </h4></a><br/> | + | <a href="#exp19"><h4> 138. Digestion of B1⁄B2, E1⁄E2 and pET43.1 (a+) with Hind III and Xba I </h4></a><br/> |
| − | <a href="#exp20"><h4> Dephosphorylation of digested pET43.1 </h4></a><br/> | + | <a href="#exp20"><h4> 139. Dephosphorylation of digested pET43.1 (a+) </h4></a><br/> |
| − | <a href="#exp21"><h4> PCR of A1⁄A2 and D1⁄D2 without MgCl<sub>2<sub> </h4></a><br/> | + | <a href="#exp21"><h4> 140. PCR of A1⁄A2 and D1⁄D2 without MgCl<sub>2<sub> </h4></a><br/> |
| − | <a href="#exp22"><h4> Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2) </h4></a><br/> | + | <a href="#exp22"><h4> 141. Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2) </h4></a><br/> |
| − | <a href="#exp23"><h4> Electrophoresis of PCR products A1⁄A2 and D1⁄D2 </h4></a><br/> | + | <a href="#exp23"><h4> 142. Electrophoresis of PCR products A1⁄A2 and D1⁄D2 </h4></a><br/> |
| − | <a href="#exp24"><h4> Next PCR of A1⁄A2 and D1⁄D2 </h4></a><br/> | + | <a href="#exp24"><h4> 143. Next PCR of A1⁄A2 and D1⁄D2 </h4></a><br/> |
| − | <a href="#exp25"><h4> Pool of inserts C1 v2 and C2 v2 </h4></a><br/> | + | <a href="#exp25"><h4> 144. Pool of inserts C1 v2 and C2 v2 </h4></a><br/> |
</p> | </p> | ||
| − | <p><B>< | + | <p><B><h3> August 5, 2016:</B></h3></p> |
<p> | <p> | ||
| − | <a href="#exp26"><h4> Digestion of B1⁄B2 and C1⁄C2 </h4></a><br/> | + | <a href="#exp26"><h4> 145. Digestion of B1⁄B2 and C1⁄C2 </h4></a><br/> |
| − | <a href="#exp27"><h4> Ligation of C1 and C2 with pET43.1 digested and dephosphorylated </h4></a><br/> | + | <a href="#exp27"><h4> 146. Ligation of C1 and C2 with pET43.1 (a+) digested and dephosphorylated </h4></a><br/> |
| − | <a href="#exp28"><h4> Transformation of TOP10 competent cells </h4></a><br/> | + | <a href="#exp28"><h4> 147. Transformation of TOP10 competent cells </h4></a><br/> |
| − | <a href= | + | <a href="#exp29"><h4> 148. Ligation of B1⁄B2⁄C1⁄C2 in pET43.1 (a+) </h4></a><br/> |
| − | <a href= | + | <a href="#exp30"><h4> 149. Transformation of B1⁄B2⁄C1⁄C2 in TOP10 </h4></a><br/> |
</p> | </p> | ||
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<p> | <p> | ||
<U> Aim:</U> Check if the sillification is working with different volume of HCl and TEOS. <br/> <br/> | <U> Aim:</U> Check if the sillification is working with different volume of HCl and TEOS. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/2/2a/Protocol-silification-assay_Pasteur_Paris2016.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 280: | Line 297: | ||
• Silification protein <br/> | • Silification protein <br/> | ||
• HCl 1 M <br/> | • HCl 1 M <br/> | ||
| − | • TEOS <br/> | + | • TEOS (Tetraethyl ortho silicate, Sigma) <br/> |
• Shaking incubator <br/> | • Shaking incubator <br/> | ||
• 15 ml Falcon <br/> | • 15 ml Falcon <br/> | ||
| − | • Buffer A <br/> | + | • Buffer A (See protein purification protocol)<br/> |
• 10 μl and 200 μl pipettes <br/><br/> | • 10 μl and 200 μl pipettes <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
| − | 1. In a 1.5 ml Eppendorf prepare a sample with | + | <li>1. In a 1.5 ml Eppendorf prepare a sample with the fraction that contains protein c2, add 1 ml of HCl 1mM, 65.8 μl of TEOS and let it incubate for 4 minutes in the rotating incubator</li> |
| − | 2. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample | + | <li>2. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample</li> |
| − | 3. Follow if silification initiates and progresses at various times : <br/> | + | <li>3. Follow if silification initiates and progresses at various times :</li> <br/> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 84</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 317: | Line 335: | ||
4. Redo this experiment with 1 ml of TEOS, 50 μl of protein and vortex: <br/> | 4. Redo this experiment with 1 ml of TEOS, 50 μl of protein and vortex: <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 85</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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5. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein : <br/> | 5. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 86</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 349: | Line 369: | ||
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 6. Redo the experiment with 50 μl of HCl and 50 μl of protein<br/> | + | 6. Redo the experiment with 50 μl of HCl 1 mM and 50 μl of protein<br/> |
| − | 7.Redo the experiment with 50 μl of HCl but without our protein<br/><br/> | + | 7.Redo the experiment with 50 μl of HCl 1 mM but without our protein<br/><br/> |
<U>Results: </U><br/> | <U>Results: </U><br/> | ||
| Line 369: | Line 389: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | + | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. |
| − | + | We also switched to the Takara EX DNA polymerase for its terminal deoxynucleotide transferase activity, which adds an deoxy-A residue at the 3' end of the PCR product<br/> <br/> | |
| + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br/><br/> | ||
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 381: | Line 402: | ||
• Takara EX DNA polymerase <br/> | • Takara EX DNA polymerase <br/> | ||
• PCR machine <br/> | • PCR machine <br/> | ||
| − | • 10 | + | • 10 μl and 200 μl pipettes <br/><br/> |
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
1. Use a Globalmix with : <br/> | 1. Use a Globalmix with : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 87</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 416: | Line 438: | ||
2. For the next step use : <br/> | 2. For the next step use : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 88</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 457: | Line 480: | ||
<tr> | <tr> | ||
<td align="center" ; valign = “center”><strong><p> 4 </p></strong></td> | <td align="center" ; valign = “center”><strong><p> 4 </p></strong></td> | ||
| − | <td align="center" ; valign = “center”> C2 insert (For +Rev) </td> | + | <td align="center" ; valign = “center”> C2 insert (For + Rev) </td> |
<td align="center" ; valign = “center”> 46.5 </td> | <td align="center" ; valign = “center”> 46.5 </td> | ||
<td align="center" ; valign = “center”> Ø </td> | <td align="center" ; valign = “center”> Ø </td> | ||
| Line 477: | Line 500: | ||
<td align="center" ; valign = “center”> C2 Rev </td> | <td align="center" ; valign = “center”> C2 Rev </td> | ||
<td align="center" ; valign = “center”> 46.5 </td> | <td align="center" ; valign = “center”> 46.5 </td> | ||
| − | <td align="center" ; valign = “center”> Ø | + | <td align="center" ; valign = “center”> Ø </td> |
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
<td align="center" ; valign = “center”> Ø </td> | <td align="center" ; valign = “center”> Ø </td> | ||
| Line 511: | Line 534: | ||
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 3. For the PCR program, start at 24°C, add 0.5 μl of Takara enzyme and proceed to 94°C | + | 3. For the PCR program, start at 24°C, add 0.5 μl of Takara enzyme and proceed to 94°C. Annealing is set at 55°C. |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 527: | Line 550: | ||
<p> | <p> | ||
<U> Aim:</U> Verification of the content of the fractions obtained from the Ni-NTA column chromatography. <br/> <br/> | <U> Aim:</U> Verification of the content of the fractions obtained from the Ni-NTA column chromatography. <br/> <br/> | ||
| − | + | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 543: | Line 566: | ||
<p> | <p> | ||
<U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 549: | Line 572: | ||
• PCR products <br/> | • PCR products <br/> | ||
• Inserts C1 v2, C2 v2 <br/> | • Inserts C1 v2, C2 v2 <br/> | ||
| − | • Primer S | + | • Primer S (For) <br/> |
| − | • Primer AS | + | • Primer AS (Rev) <br/> |
• Electrophoresis chamber, and power supply <br/> | • Electrophoresis chamber, and power supply <br/> | ||
• 10 μl pipette <br/><br/> | • 10 μl pipette <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
1. Add 5 μl of DNA and 1 μl of buffer 6X to each sample | 1. Add 5 μl of DNA and 1 μl of buffer 6X to each sample | ||
| − | 2. Use 6 | + | 2. Use 6 μl of molecular weight marker and deposit each sample in the wells :<br/> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 89</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 589: | Line 613: | ||
</table><br/> | </table><br/> | ||
2. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A <br/><br/> | 2. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A <br/><br/> | ||
| − | |||
| − | |||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 605: | Line 627: | ||
<p> | <p> | ||
<U> Aim:</U> Get back the DNA amplified by PCR. <br/> <br/> | <U> Aim:</U> Get back the DNA amplified by PCR. <br/> <br/> | ||
| − | |||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 620: | Line 641: | ||
<p> | <p> | ||
<U> Aim:</U> Increase the quantity of DNA for cloning. <br/> <br/> | <U> Aim:</U> Increase the quantity of DNA for cloning. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br/><br/> | |
| + | <U> Results :</U><br/> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/9/9b/Week_9_Figure_2_Gel_de_la_PCR_du_01-08-16_A1_A2_B1_B2_D1_D2_E1_E2.jpg"; alt = "PCR gel of A1_A2_B1_B2_D1_D2_E1_E2"/> | ||
| + | <i><p><U>Figure 11 :</U> PCR gel of A1/A2/B1/B2/D1/D2/E1/E2 </p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 635: | Line 659: | ||
<p> | <p> | ||
<U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> <U>What we did in the lab:</U><br/> | |
| − | + | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
• Agarose <br/> | • Agarose <br/> | ||
| − | • Ethidium bromide | + | • Ethidium bromide drops (EB) <br/> |
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher) <br/> | • Molecular weight ladder (Gene ruler 1 Kb, Thermofisher) <br/> | ||
• Loading buffer 6X <br/> | • Loading buffer 6X <br/> | ||
| Line 646: | Line 669: | ||
• 10 μl pipette <br/><br/> | • 10 μl pipette <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
| − | 1. Make an agarose gel at 0.7 | + | 1. Make an agarose gel at 0.7% |
| − | 2. Prepare the samples with 5 | + | 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X |
3. Depose each samples in the wells : <br/> | 3. Depose each samples in the wells : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 90</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 692: | Line 716: | ||
<U>Result</U><br/> | <U>Result</U><br/> | ||
There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14. <br/> | There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14. <br/> | ||
| − | The PCR must be done another time for A1⁄A2 and D11#8260;D2 with a lower temperature (1°C). | + | The PCR must be done another time for A1⁄A2 and D11⁄D2 with a lower temperature (1°C). |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 707: | Line 731: | ||
<p> | <p> | ||
<U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | ||
| − | |||
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 716: | Line 739: | ||
1. Prepare the 8 samples: <br/> | 1. Prepare the 8 samples: <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 91</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 745: | Line 769: | ||
2. Follow the protocol of the Qiagen PCR clean up kit <br/><br/> | 2. Follow the protocol of the Qiagen PCR clean up kit <br/><br/> | ||
<U>Result</U><br/> | <U>Result</U><br/> | ||
| − | We obtained 25 | + | We obtained 25 μl of each insert purified by PCR. |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 761: | Line 785: | ||
<p> | <p> | ||
<U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 773: | Line 797: | ||
1 For each inserts, prepare the following mix : <br/> | 1 For each inserts, prepare the following mix : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 92</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 815: | Line 840: | ||
<p> | <p> | ||
<U> Aim:</U> Check if our plasmid was inserted in the bacteria, and to recover larger amounts of it. <br/> <br/> | <U> Aim:</U> Check if our plasmid was inserted in the bacteria, and to recover larger amounts of it. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br/><br/> | |
| − | < | + | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 830: | Line 855: | ||
<p> | <p> | ||
<U> Aim:</U> Increase the quality of DNA. <br/> <br/> | <U> Aim:</U> Increase the quality of DNA. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| − | • | + | • dNTPs <br/> |
• MgCl<sub>2</sub> <br/> | • MgCl<sub>2</sub> <br/> | ||
• Buffer 20X <br/> | • Buffer 20X <br/> | ||
| Line 846: | Line 871: | ||
1. Prepare a Globalmix with : <br/> | 1. Prepare a Globalmix with : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 93</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 854: | Line 880: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> dNTPs </p></strong></td> |
<td align="center" ; valign = “center”> 12.5</td> | <td align="center" ; valign = “center”> 12.5</td> | ||
</tr> | </tr> | ||
| Line 885: | Line 911: | ||
2. Prepare the following samples : | 2. Prepare the following samples : | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 94</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 896: | Line 923: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> <strong><p> V<sub>inserts</sub> (μl) </p></strong></td> | + | <td align="center" ; valign = “center”><strong><p> V<sub>inserts</sub> (μl) </p></strong></td> |
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
| Line 927: | Line 954: | ||
<p> | <p> | ||
<U> Aim:</U> Check the efficiency of the PCR. <br/> <br/> | <U> Aim:</U> Check the efficiency of the PCR. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| Line 935: | Line 962: | ||
• Loading buffer 6X <br/> | • Loading buffer 6X <br/> | ||
• PCR products <br/> | • PCR products <br/> | ||
| − | • 10 &# | + | • 10 μl pipette <br/> |
• Electrophoresis machine <br/> | • Electrophoresis machine <br/> | ||
• 1.5 ml Eppendorfs <br/><br/> | • 1.5 ml Eppendorfs <br/><br/> | ||
| Line 943: | Line 970: | ||
3. Deposit each sample in the wells : <br/> | 3. Deposit each sample in the wells : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 95</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 968: | Line 996: | ||
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30 | + | 4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30. |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 984: | Line 1,012: | ||
<p> | <p> | ||
<U> Aim:</U> Get DNA back. <br/> <br/> | <U> Aim:</U> Get DNA back. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link | + | <U> Protocol:</U> follow in this link <br/><br/> |
| + | <U>Results</U><br/> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/0/04/Week_9_Figure_3_gel_miniprep_topoclonning_C1_de_1_à_8_digéré_XbaI-HindIII.jpg"; alt = "Digestion gel of C1 (1-8)"/> | ||
| + | <i><p><U>Figure 12 :</U> Digestion gel of C1 (1-8)</p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 998: | Line 1,029: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <U> Aim:</U> Recombine the inserts with the plasmid. <br/> <br/> |
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials:</U><br/> | <U>Materials:</U><br/> | ||
| − | • Hind III (NEB) | + | • Hind III (NEB) <br/> |
| − | • Xba I 5NEB) | + | • Xba I 5NEB) <br/> |
| − | • Buffer CutSmart 10X (NEB) | + | • Buffer CutSmart 10X (NEB) <br/> |
| − | • H<sub>2</sub>O | + | • H<sub>2</sub>O <br/> |
| − | • 1.5 Eppendorfs | + | • 1.5 Eppendorfs <br/> |
| − | • C1 v2 and C2 v2 | + | • C1 v2 and C2 v2 <br/> |
| − | • Incubator 37°C | + | • Incubator 37°C <br/> |
• 20 μl and 200 μl pipettes <br/><br/> | • 20 μl and 200 μl pipettes <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
1. Prepare a Globalmix : <br/> | 1. Prepare a Globalmix : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 96</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,049: | Line 1,081: | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| + | |||
<div class="lightbox" id="exp15"> | <div class="lightbox" id="exp15"> | ||
| Line 1,056: | Line 1,090: | ||
<p> | <p> | ||
<U> Aim:</U> Check if our inserts have been correctly digested by the enzyme. <br/> <br/> | <U> Aim:</U> Check if our inserts have been correctly digested by the enzyme. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
Lane 1 : <br/> | Lane 1 : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 97</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,124: | Line 1,159: | ||
Lane 2: <br/> | Lane 2: <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 98</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,199: | Line 1,235: | ||
<p> | <p> | ||
<U> Aim:</U> Get back the DNA which was digested and purified. <br/> <br/> | <U> Aim:</U> Get back the DNA which was digested and purified. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,207: | Line 1,243: | ||
<U>Results</U><br/> | <U>Results</U><br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 99</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,215: | Line 1,252: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.2 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.2 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3435 </td> | <td align="center" ; valign = “center”> 1.3435 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.4 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.4 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3564 </td> | <td align="center" ; valign = “center”> 1.3564 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.5 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.5 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3825 </td> | <td align="center" ; valign = “center”> 1.3825 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.6 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.6 </p></strong></td> |
<td align="center" ; valign = “center”> 1.4318 </td> | <td align="center" ; valign = “center”> 1.4318 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.7 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.7 </p></strong></td> |
<td align="center" ; valign = “center”> 1.4392 </td> | <td align="center" ; valign = “center”> 1.4392 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.8 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.8 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3564 </td> | <td align="center" ; valign = “center”> 1.3564 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C1.10 </td> | + | <td align="center" ; valign = “center”><strong><p> C1.10 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3800 </td> | <td align="center" ; valign = “center”> 1.3800 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C2.1 </td> | + | <td align="center" ; valign = “center”><strong><p> C2.1 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3179 </td> | <td align="center" ; valign = “center”> 1.3179 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C2.2 </td> | + | <td align="center" ; valign = “center”><strong><p> C2.2 </p></strong></td> |
<td align="center" ; valign = “center”> 1.2500 </td> | <td align="center" ; valign = “center”> 1.2500 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C2.3 </td> | + | <td align="center" ; valign = “center”><strong><p> C2.3 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3910 </td> | <td align="center" ; valign = “center”> 1.3910 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C2.9 </td> | + | <td align="center" ; valign = “center”><strong><p> C2.9 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3668 </td> | <td align="center" ; valign = “center”> 1.3668 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> C2.10 </td> | + | <td align="center" ; valign = “center”><strong><p> C2.10 </p></strong></td> |
<td align="center" ; valign = “center”> 1.3934 </td> | <td align="center" ; valign = “center”> 1.3934 </td> | ||
</tr> | </tr> | ||
| Line 1,278: | Line 1,315: | ||
<p> | <p> | ||
<U> Aim:</U> Put the insert into the plasmid and transform the plasmid into bacteria. <br/> <br/> | <U> Aim:</U> Put the insert into the plasmid and transform the plasmid into bacteria. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br/><br/> |
<U>Results (on the August 4, 2016)</U><br/> | <U>Results (on the August 4, 2016)</U><br/> | ||
Several colonies have grown but they are too small so we let them grow more. But C1.2 has much more colonies so we spread it into another Petri dish. <br/><br/><br/> | Several colonies have grown but they are too small so we let them grow more. But C1.2 has much more colonies so we spread it into another Petri dish. <br/><br/><br/> | ||
| Line 1,294: | Line 1,331: | ||
<p> | <p> | ||
<U> Aim:</U> Get back the DNA from bacteria. <br/> <br/> | <U> Aim:</U> Get back the DNA from bacteria. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br/><br/> | |
| + | <U> Results </U><br/> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/2/2b/Week_9_Figure_4_gel_miniprep_topocloning_de_B1_et_B2_colonies_1_a_10_digéré_X%2BH_05-05-16.jpg"; alt = "Digestion gel of B1/B2 (1-10)"/> | ||
| + | <i><p><U>Figure 13 :</U> Digestion gel of B1/B2 (1-10)</p></i></center> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/c/cf/Week_9_Figure_5_gel_miniprep_topocloning_de_E1_et_E2_colonies_1_a_10_digéré_X%2BH_05-05-16.jpg"; alt = "Digestion gel of E1/E2 (1-10)"/> | ||
| + | <i><p><U>Figure 14 :</U> Digestion gel of E1/E2 (1-10)</p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 1,309: | Line 1,351: | ||
<p> | <p> | ||
<U> Aim:</U> Prepare the ligation. <br/> <br/> | <U> Aim:</U> Prepare the ligation. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| − | • 1.5 ml Eppendorfs | + | • 1.5 ml Eppendorfs <br/> |
| − | • DNA | + | • DNA <br/> |
| − | • Buffer CutSmart (NEB) | + | • Buffer CutSmart (NEB) <br/> |
| − | • HindIII (NEB) | + | • HindIII (NEB) <br/> |
| − | • XbaI (NEB) | + | • XbaI (NEB) <br/> |
| − | • H<sub>2</sub>O | + | • H<sub>2</sub>O <br/> |
| − | • Samples B1⁄B2, E1⁄E2 | + | • Samples B1⁄B2, E1⁄E2 <br/> |
• 10 μl and 20 μl pipettes <br/><br/> | • 10 μl and 20 μl pipettes <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
1. Make a master mix with : <br/> | 1. Make a master mix with : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 100</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,378: | Line 1,421: | ||
<p> | <p> | ||
<U> Aim:</U> Avoid the religation of the plasmid with itself. <br/> <br/> | <U> Aim:</U> Avoid the religation of the plasmid with itself. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> 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/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| − | • 1.5 ml Eppendorfs | + | • 1.5 ml Eppendorfs <br/> |
| − | • Digested DNA | + | • Digested DNA <br/> |
| − | • Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB) | + | • Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB) <br/> |
| − | • Buffer CutSmart | + | • Buffer CutSmart <br/> |
| − | • H<sub>2</sub>O | + | • H<sub>2</sub>O <br/> |
| − | • Incubator 37°C | + | • Incubator 37°C <br/> |
• 10 μl and 20 μl pipettes | • 10 μl and 20 μl pipettes | ||
<br/><br/> | <br/><br/> | ||
| Line 1,392: | Line 1,435: | ||
1. In a 1.5 ml Eppendorf, put : <br/> | 1. In a 1.5 ml Eppendorf, put : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 101</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,437: | Line 1,481: | ||
<p> | <p> | ||
<U> Aim:</U> Increase the quantity of DNA. <br/> <br/> | <U> Aim:</U> Increase the quantity of DNA. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,455: | Line 1,499: | ||
1. Prepare the mix : <br/> | 1. Prepare the mix : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 102</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,463: | Line 1,508: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> dNTPs </p></strong></td> |
<td align="center" ; valign = “center”> 12.5 </td> | <td align="center" ; valign = “center”> 12.5 </td> | ||
</tr> | </tr> | ||
| Line 1,479: | Line 1,524: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> Primer S </p></strong></td> | + | <td align="center" ; valign = “center”><strong><p> Primer S (For) </p></strong></td> |
<td align="center" ; valign = “center”> 5 </td> | <td align="center" ; valign = “center”> 5 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> Primer AS </p></strong></td> | + | <td align="center" ; valign = “center”><strong><p> Primer AS(Rev) </p></strong></td> |
<td align="center" ; valign = “center”> 5 </td> | <td align="center" ; valign = “center”> 5 </td> | ||
</tr> | </tr> | ||
| Line 1,494: | Line 1,539: | ||
2. To put the sample into the PCR machine, use the table : <br/> | 2. To put the sample into the PCR machine, use the table : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 103</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,544: | Line 1,590: | ||
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 3. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 μl of Takara enzyme in each tube | + | 3. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 μl of Takara enzyme in each tube<br/><br/> |
| + | <U>Results</U><br/> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/c/ca/Week_9_Figure_6_PCR_A1_A2_D1_D2_sans_MgCl2_04-08-16.jpg"; alt = "PCR gel of A1/A2/D1/D2 without MGCl<sub>2</sub>"/> | ||
| + | <i><p><U>Figure 15 :</U> PCR gel of A1/A2/D1/D2 without MGCl<sub>2</sub> </p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 1,559: | Line 1,608: | ||
<p> | <p> | ||
<U> Aim:</U> Have more bacteria with the insert A. <br/> <br/> | <U> Aim:</U> Have more bacteria with the insert A. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,590: | Line 1,639: | ||
<p> | <p> | ||
<U> Aim:</U> Check if the PCR is efficient. <br/> <br/> | <U> Aim:</U> Check if the PCR is efficient. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,602: | Line 1,651: | ||
1. Make a 0.7% agarose gel <br/> | 1. Make a 0.7% agarose gel <br/> | ||
2. Add 5 μl of DNA and 1 μl of loading buffer. But we used 10 μl of DNA for the sample 4 <br/> | 2. Add 5 μl of DNA and 1 μl of loading buffer. But we used 10 μl of DNA for the sample 4 <br/> | ||
| − | 3. | + | 3. Deposit the sample for the electrophoresis according to this: <br/> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 104</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,647: | Line 1,697: | ||
<p> | <p> | ||
<U> Aim:</U> Increase the quantity of DNA. <br/> <br/> | <U> Aim:</U> Increase the quantity of DNA. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,662: | Line 1,712: | ||
1. Make a Globalmix with : <br/> | 1. Make a Globalmix with : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 105</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,670: | Line 1,721: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> dNTPs </p></strong></td> |
<td align="center" ; valign = “center”> 72.5 </td> | <td align="center" ; valign = “center”> 72.5 </td> | ||
</tr> | </tr> | ||
| Line 1,701: | Line 1,752: | ||
2. Prepare the samples : <br/> | 2. Prepare the samples : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 106</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,738: | Line 1,790: | ||
3. Deposit the samples on the gel: <br/> | 3. Deposit the samples on the gel: <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 107</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,799: | Line 1,852: | ||
</tbody> | </tbody> | ||
</table><br/> | </table><br/> | ||
| − | 4. Make a 0.7% agarose gel and perform an electrophoresis with 5 | + | 4. Make a 0.7% agarose gel and perform an electrophoresis with 5 µl of DNA and 1 µl of loading 6X buffer for each sample <br/><br/> |
<U>Results</U><br/> | <U>Results</U><br/> | ||
| − | <img | + | <center><img scr = "https://static.igem.org/mediawiki/2016/5/51/Week_9_Figure_7_Gel_de_la_PCR_A1_A2_B1_B2_D1_D2_E1_E2.jpg"; alt = "PCR gel of A1/A2/B1/B2/D1/D2/E1/E2"/> |
| + | <i><p><U>Figure 16 :</U> PCR gel of A1/A2/B1/B2/D1/D2/E1/E2 </p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 1,816: | Line 1,870: | ||
<p> | <p> | ||
<U> Aim:</U> Increase the DNA. <br/> <br/> | <U> Aim:</U> Increase the DNA. <br/> <br/> | ||
| − | |||
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,833: | Line 1,886: | ||
 For C1 : 35 μl of NaOAc and 875 μl of ethanol <br/> |  For C1 : 35 μl of NaOAc and 875 μl of ethanol <br/> | ||
 For C2 : 25 μl of NaOAc and 625 μl of ethanol <br/> |  For C2 : 25 μl of NaOAc and 625 μl of ethanol <br/> | ||
| − | 3. Mix the samples and incubate | + | 3. Mix the samples and incubate 8 minutes at -80°C <br/> |
| − | 4. Centrifuge 5 minutes at 4°C and 15000 RPM. | + | 4. Centrifuge 5 minutes at 4°C and 15000 RPM. Then, throw out the supernatant <br/> |
5. Add 1 ml of ethanol ice cold (70%) <br/> | 5. Add 1 ml of ethanol ice cold (70%) <br/> | ||
6. Centrifuge 15 minutes at 4°C and 15000 RPM <br/> | 6. Centrifuge 15 minutes at 4°C and 15000 RPM <br/> | ||
7. Throw out the supernatant and let dry at room temperature <br/> | 7. Throw out the supernatant and let dry at room temperature <br/> | ||
| − | 8. Resuspend the pellet in 50 μl of buffer E | + | 8. Resuspend the pellet in 50 μl of buffer E.<br/><br/> |
| + | <U>Results</U><br/> | ||
| + | <center><img scr = "https://static.igem.org/mediawiki/2016/f/ff/Week_9_Figure_8_PCR_C1_v2_C2_v2_TakaraEx_01-08-2016.jpg"; alt = "PCR gel of C1 v2 / C2 v2 with Takara enzyme"/> | ||
| + | <i><p><U>Figure 17 :</U> PCR gel of C1 v2 / C2 v2 with Takara enzyme </p></i></center> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 1,852: | Line 1,908: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Make the inserts | + | <U> Aim:</U> Make the inserts ligate with the plasmid. <br/> <br/> |
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
• 10 μl, 20 μl and 200 μl pipette <br/> | • 10 μl, 20 μl and 200 μl pipette <br/> | ||
• Hind III (NEB) <br/> | • Hind III (NEB) <br/> | ||
| − | • | + | • Xba I (NEB) <br/> |
• Buffer CutSmart <br/> | • Buffer CutSmart <br/> | ||
• H<sub>2</sub>O <br/> | • H<sub>2</sub>O <br/> | ||
| Line 1,866: | Line 1,922: | ||
1. Make a Globalmix with : <br/> | 1. Make a Globalmix with : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 108</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,874: | Line 1,931: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> Hind III </p></strong></td> |
<td align="center" ; valign = “center”> 45 </td> | <td align="center" ; valign = “center”> 45 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> Xba I </p></strong></td> |
<td align="center" ; valign = “center”> 45 </td> | <td align="center" ; valign = “center”> 45 </td> | ||
</tr> | </tr> | ||
| Line 1,897: | Line 1,954: | ||
2. Distribute this global mix in each of our 4 tubes so they will contain : <br/> | 2. Distribute this global mix in each of our 4 tubes so they will contain : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 109</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,909: | Line 1,967: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> Hind III </p></strong></td> |
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> Xb aI </p></strong></td> |
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
</tr> | </tr> | ||
| Line 1,946: | Line 2,004: | ||
<p> | <p> | ||
<U> Aim:</U> Prepare the plasmid for the transformation. <br/> <br/> | <U> Aim:</U> Prepare the plasmid for the transformation. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br/><br/> |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 1,955: | Line 2,013: | ||
• H<sub>2</sub>O <br/> | • H<sub>2</sub>O <br/> | ||
• Incubator <br/> | • Incubator <br/> | ||
| − | • 10 | + | • 10 μl and 20 μl pipettes |
<br/><br/> | <br/><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
1. In a 1.5 ml eppendorf, put : <br/> | 1. In a 1.5 ml eppendorf, put : <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 110</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,965: | Line 2,024: | ||
<th> C1 </th> | <th> C1 </th> | ||
<th> C2 </th> | <th> C2 </th> | ||
| − | <th> pET 43.1 alone </th> | + | <th> pET 43.1 (a+) alone </th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 1,981: | Line 2,040: | ||
<td align="center" ; valign = “center”> Ø </td> | <td align="center" ; valign = “center”> Ø </td> | ||
</tr> | </tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> pET 43. | + | <td align="center" ; valign = “center”><strong><p> pET 43.1a(+) (μl) </p></strong></td> |
<td align="center" ; valign = “center”> 4 </td> | <td align="center" ; valign = “center”> 4 </td> | ||
<td align="center" ; valign = “center”> 4 </td> | <td align="center" ; valign = “center”> 4 </td> | ||
<td align="center" ; valign = “center”> 4 </td> | <td align="center" ; valign = “center”> 4 </td> | ||
</tr> | </tr> | ||
| − | <td align="center" ; valign = “center”><strong><p> | + | <td align="center" ; valign = “center”><strong><p> T4 ligase (μl) </p></strong></td> |
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
<td align="center" ; valign = “center”> 1 </td> | <td align="center" ; valign = “center”> 1 </td> | ||
| Line 2,025: | Line 2,084: | ||
<p> | <p> | ||
<U> Aim:</U> Express our plasmid in bacteria. <br/> <br/> | <U> Aim:</U> Express our plasmid in bacteria. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> 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/> | <U>What we did in the lab:</U><br/> | ||
<U>Materials</U><br/> | <U>Materials</U><br/> | ||
| Line 2,033: | Line 2,092: | ||
Use the Qiagen gel extraction kit for a final volume of 50 μl. <br/> | Use the Qiagen gel extraction kit for a final volume of 50 μl. <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 111</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 2,041: | Line 2,101: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> B1 colony 1 </td> | + | <td align="center" ; valign = “center”><strong><p> B1 colony 1 </p></strong></td> |
<td align="center" ; valign = “center”> 136 </td> | <td align="center" ; valign = “center”> 136 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> B1 colony 2 </td> | + | <td align="center" ; valign = “center”><strong><p> B1 colony 2 </p></strong></td> |
<td align="center" ; valign = “center”> 170 </td> | <td align="center" ; valign = “center”> 170 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> B2 colony 4 </td> | + | <td align="center" ; valign = “center”><strong><p> B2 colony 4 </p></strong></td> |
<td align="center" ; valign = “center”> 166 </td> | <td align="center" ; valign = “center”> 166 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> B2 colony 7 </td> | + | <td align="center" ; valign = “center”><strong><p> B2 colony 7 </strong></p></td> |
<td align="center" ; valign = “center”> 175 </td> | <td align="center" ; valign = “center”> 175 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> E1 colony 1 </td> | + | <td align="center" ; valign = “center”><strong><p> E1 colony 1 </strong></p></td> |
<td align="center" ; valign = “center”> 110 </td> | <td align="center" ; valign = “center”> 110 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> E1 colony 2 </td> | + | <td align="center" ; valign = “center”><strong><p> E1 colony 2 </strong></p></td> |
<td align="center" ; valign = “center”> 143 </td> | <td align="center" ; valign = “center”> 143 </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center" ; valign = “center”> E2 colony 1 </td> | + | <td align="center" ; valign = “center”><strong><p> E2 colony 1 </p></strong></td> |
<td align="center" ; valign = “center”> 108 </td> | <td align="center" ; valign = “center”> 108 </td> | ||
</tr> | </tr> | ||
| Line 2,088: | Line 2,148: | ||
<p> | <p> | ||
<U> Aim:</U> Ligate our inserts with the plasmid. <br/> <br/> | <U> Aim:</U> Ligate our inserts with the plasmid. <br/> <br/> | ||
| − | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br/><br/> | |
<U>What we did in the lab:</U><br/> | <U>What we did in the lab:</U><br/> | ||
<U>Method:</U><br/> | <U>Method:</U><br/> | ||
We use the next volumes: <br/> | We use the next volumes: <br/> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 112</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 2,136: | Line 2,197: | ||
<p> | <p> | ||
<U> Aim:</U> Put our ligated plasmid into bacteria. <br/> <br/> | <U> Aim:</U> Put our ligated plasmid into bacteria. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this link<br/><br/> | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br/><br/> |
<U>Results</U><br/> | <U>Results</U><br/> | ||
48 hours later, no colony had grown so the transformation did not work, it has to be redone. | 48 hours later, no colony had grown so the transformation did not work, it has to be redone. | ||
| Line 2,145: | Line 2,206: | ||
</div> | </div> | ||
</body> | </body> | ||
| − | |||
</html> | </html> | ||
