Difference between revisions of "Team:Pasteur Paris/Microbiology week10"
| Line 272: | Line 272: | ||
<p><B><h3> August 12, 2016:</B></h3></p> | <p><B><h3> August 12, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp34"><h4> 183. Miniprep from precultures of B2 v2 | + | <a href="#exp34"><h4> 183. Miniprep from precultures of B2 v2/E1/E2 in TOPO </h4></a><br/> |
| − | <a href="#exp35"><h4> 184. Digestion of inserts B2 v2 | + | <a href="#exp35"><h4> 184. Digestion of inserts B2 v2/E1/E2 with XbaI and HindIII </h4></a><br/> |
<a href="#exp36"><h4> 185. Culture of C2 v2 and B1 v2 in 1 l of LB </h4></a><br/> | <a href="#exp36"><h4> 185. Culture of C2 v2 and B1 v2 in 1 l of LB </h4></a><br/> | ||
| − | <a href="#exp37"><h4> 186. Agarose gel to analyze digestion of pET 43.1 (a+) done on the 11<sup>th</sup> of August </h4></a><br/> | + | <a href="#exp37"><h4> 186. Agarose gel to analyze digestion of pET 43.1(a+) done on the 11<sup>th</sup> of August </h4></a><br/> |
</p> | </p> | ||
| Line 293: | Line 293: | ||
• Swing bucket centrifuge (JOUAN GR41)<br /> | • Swing bucket centrifuge (JOUAN GR41)<br /> | ||
• Colonies of C2 v2 and B1 v2 <br /> | • Colonies of C2 v2 and B1 v2 <br /> | ||
| − | • carbenicillin at 50 mg | + | • carbenicillin at 50 mg/ml <br /> |
• LB medium <br /> | • LB medium <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>) | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) | ||
<br /><br /> | <br /><br /> | ||
<h6><U>Method :</U></h6> | <h6><U>Method :</U></h6> | ||
| − | 1. In a 50 ml falcon, put 48 ml of LB and 48 &# | + | 1. In a 50 ml falcon, put 48 ml of LB and 48 µl of carbenicillin. <br /> |
2. For B1 v2 : <br /> | 2. For B1 v2 : <br /> | ||
  2.a Prepare 13 Eppendorfs of 1.5 ml in which add 1 ml of the previous mix. <br /> |   2.a Prepare 13 Eppendorfs of 1.5 ml in which add 1 ml of the previous mix. <br /> | ||
  2.b Take with a toothpick colonies on the petri dish. <br /> |   2.b Take with a toothpick colonies on the petri dish. <br /> | ||
  2.c Place the toothpick in a tube. <br /> |   2.c Place the toothpick in a tube. <br /> | ||
| − |   2.d Let incubate overnight at | + |   2.d Let incubate overnight at 37°C and 150 rpm. <br /> |
3. For C2 v2 : <br /> | 3. For C2 v2 : <br /> | ||
  3.a Prepare 20 eppendorfs of 1.5 ml with 1 ml of LB and carbenicillin mix.<br /> |   3.a Prepare 20 eppendorfs of 1.5 ml with 1 ml of LB and carbenicillin mix.<br /> | ||
| Line 395: | Line 395: | ||
Ladder | Ø | B2 (1) | B2 (2) | B2 (3) | B2 (4) | B2 (5) | B2 (6) | B2 (7) | B2 (8) | B2 (9) | B2 (10) | Ø | E1 (1) | E1 (2) | E1 (3) | E1 (4) | E1 (5) | Ø | Ø | ladder | E1 (6) | E1 (7) | E1 (8) | E1 (9) | E1 (10) | Ø | E2 (1) | E2 (2) | E2 (3) | E2 (4) | E2 (5) | E2 (6) | E2 (7) | E2 (8) | E2 (9) | E2 (10) | Ø | Ø <br/> | Ladder | Ø | B2 (1) | B2 (2) | B2 (3) | B2 (4) | B2 (5) | B2 (6) | B2 (7) | B2 (8) | B2 (9) | B2 (10) | Ø | E1 (1) | E1 (2) | E1 (3) | E1 (4) | E1 (5) | Ø | Ø | ladder | E1 (6) | E1 (7) | E1 (8) | E1 (9) | E1 (10) | Ø | E2 (1) | E2 (2) | E2 (3) | E2 (4) | E2 (5) | E2 (6) | E2 (7) | E2 (8) | E2 (9) | E2 (10) | Ø | Ø <br/> | ||
4. Launch the electrophoresis for 20 minutes at 50 V, then 45 minutes at 130 V. <br /><br /> | 4. Launch the electrophoresis for 20 minutes at 50 V, then 45 minutes at 130 V. <br /><br /> | ||
| − | <h6><U>Results :</U></h6> | + | <h6><U>Results :</U></h6><br /><br /> |
<center><img src = "https://static.igem.org/mediawiki/2016/c/cf/Week_10_152Electrophoresis_with_the_results_of_digestion.jpg"; alt "Gel of the results of digestion"/> | <center><img src = "https://static.igem.org/mediawiki/2016/c/cf/Week_10_152Electrophoresis_with_the_results_of_digestion.jpg"; alt "Gel of the results of digestion"/> | ||
<i><p> <U>Figure 1:</U> Gel of the results of digestion </i></p></center><br /> | <i><p> <U>Figure 1:</U> Gel of the results of digestion </i></p></center><br /> | ||
| Line 553: | Line 553: | ||
  D1 : m = 120 mg<br /> |   D1 : m = 120 mg<br /> | ||
  D2 : m = 152 mg<br /><br /> |   D2 : m = 152 mg<br /><br /> | ||
| − | <h6><U>Results :</U></h6> | + | <h6><U>Results :</U></h6><br /><br /> |
<center><img src = "https://static.igem.org/mediawiki/2016/b/bb/Week_10_154Gel_extraction_of_A1-A2-D1-D2.jpg"; alt "Extraction gel of A1-A2-D1-D2"/> | <center><img src = "https://static.igem.org/mediawiki/2016/b/bb/Week_10_154Gel_extraction_of_A1-A2-D1-D2.jpg"; alt "Extraction gel of A1-A2-D1-D2"/> | ||
| − | <i><p> Figure 3 : Extraction gel of A1-A2-D1-D2 </p></i></center> | + | <i><p> <U>Figure 3 :</U> Extraction gel of A1-A2-D1-D2 </p></i></center> |
<br /><br /> <br /> | <br /><br /> <br /> | ||
</p> | </p> | ||
| Line 561: | Line 561: | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| Line 573: | Line 574: | ||
<h6><U>What we did in the lab:</U></h6><br /> | <h6><U>What we did in the lab:</U></h6><br /> | ||
<h6><U>Materials:</U></h6> | <h6><U>Materials:</U></h6> | ||
| − | • Gel of B2 | + | • Gel of B2/E1/E2 <br /> |
• QIAGEN Extraction gel kit<br /><br /> | • QIAGEN Extraction gel kit<br /><br /> | ||
<h6><U>Method:</U></h6> | <h6><U>Method:</U></h6> | ||
| Line 659: | Line 660: | ||
<h6><U>Materials :</U></h6> | <h6><U>Materials :</U></h6> | ||
• NaAc <br /> | • NaAc <br /> | ||
| − | • Ethanol 70 | + | • Ethanol 70%; <br /> |
| − | • Inserts B2 | + | • Inserts B2/E1/E2 <br /><br /> |
<h6><U>Method :</U></h6> | <h6><U>Method :</U></h6> | ||
1. Use 1/10 volume of NaAc and 2.5 colume of ethanol for each insert : <br /><br /> | 1. Use 1/10 volume of NaAc and 2.5 colume of ethanol for each insert : <br /><br /> | ||
| Line 739: | Line 740: | ||
<td align="center"; valign="center"> 15 </td> | <td align="center"; valign="center"> 15 </td> | ||
<td align="center" ; valign="center"> Ø </td> | <td align="center" ; valign="center"> Ø </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> E2 (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> E2 (µl) </p></strong></td> | ||
<td align="center"; valign="center"> Ø </td> | <td align="center"; valign="center"> Ø </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> 15 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> B2 (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> B2 (µl) </p></strong></td> | ||
<td align="center"; valign="center"> Ø </td> | <td align="center"; valign="center"> Ø </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 15 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> pET 43.1(a+) (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> pET 43.1(a+) (µl) </p></strong></td> | ||
<td align="center"; valign="center"> 4 </td> | <td align="center"; valign="center"> 4 </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> 4 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 4 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 4 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> Ligase (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> Ligase (µl) </p></strong></td> | ||
<td align="center"; valign="center"> 1 </td> | <td align="center"; valign="center"> 1 </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> 1 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 1 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 1 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> TOP0 (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> TOP0 (µl) </p></strong></td> | ||
<td align="center"; valign="center"> 2.2 </td> | <td align="center"; valign="center"> 2.2 </td> | ||
| − | <td align = | + | <td align = "center"; valign="center"> 2.2 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 2.2 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 2.2 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td align="center"; valign="center"><strong><p> H<sub>2</sub>O (µl) </p></strong></td> | <td align="center"; valign="center"><strong><p> H<sub>2</sub>O (µl) </p></strong></td> | ||
| − | <td align="center"; valign="center" | + | <td align="center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> Ø </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 15 </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td align="center"; valign="center" | + | <td align="center"; valign="center"><strong><p> V<sub>total</sub> (µl) </p></strong></td> |
| − | <td align="center"; valign="center" | + | <td align="center"; valign="center"> 22.2 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 22.2 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 22.2 </td> |
| − | <td align = | + | <td align = "center"; valign="center"> 22.2 </td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
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<h6><U>Method :</U></h6> | <h6><U>Method :</U></h6> | ||
1. Use the Qiagen kit for our cultures from the 8<sup>th</sup> of August : <br /> | 1. Use the Qiagen kit for our cultures from the 8<sup>th</sup> of August : <br /> | ||
| − |   13 Eppendorfs of B1. <br /> | + |   13 Eppendorfs of B1 v2. <br /> |
| − |   20 Eppendorfs of C2. <br /> | + |   20 Eppendorfs of C2 v2. <br /> |
2. Digest the plasmid with the following volumes for each sample : <br /> | 2. Digest the plasmid with the following volumes for each sample : <br /> | ||
<table> | <table> | ||
| Line 890: | Line 891: | ||
• C1 v2 colonies <br /> | • C1 v2 colonies <br /> | ||
• Shaking incubator (INFORS HT)<br /><br /> | • Shaking incubator (INFORS HT)<br /><br /> | ||
| − | <h6><U>Method:</U></h6> | + | <h6><U>Method :</U></h6> |
1. Prepare 20 ml of LB with 20 µl of carbenicillin. <br /> | 1. Prepare 20 ml of LB with 20 µl of carbenicillin. <br /> | ||
2. Put 1 ml of this mix in twenty 1.5 ml Eppendorfs. <br /> | 2. Put 1 ml of this mix in twenty 1.5 ml Eppendorfs. <br /> | ||
| Line 910: | Line 911: | ||
<p> | <p> | ||
| − | <h6><U> Aim:</U></h6> Create a stock of antibiotic. <br /> | + | <h6><U> Aim :</U></h6> Create a stock of antibiotic. <br /> |
| − | <h6><U>What we did in the lab:</U></h6><br /> | + | <h6><U>What we did in the lab :</U></h6><br /> |
| − | <h6><U>Materials:</U></h6> | + | <h6><U>Materials :</U></h6> |
• 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 /> | • 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 /> | ||
• Carbenicillin at 50 mg/ml <br /> | • Carbenicillin at 50 mg/ml <br /> | ||
| Line 918: | Line 919: | ||
• 15 ml Falcon <br /> | • 15 ml Falcon <br /> | ||
• Distilled water <br /><br /> | • Distilled water <br /><br /> | ||
| − | <h6><U>Method:</U></h6> | + | <h6><U>Method :</U></h6> |
1. Prep: Put 500 mg of carbenicillin in a 15 ml Falcon and 10 ml of distilled water. Then, put the Falcon on ice. <br /> | 1. Prep: Put 500 mg of carbenicillin in a 15 ml Falcon and 10 ml of distilled water. Then, put the Falcon on ice. <br /> | ||
2. Aliquot the mix in 10 Eppendorfs of 1.5 ml. <br /> | 2. Aliquot the mix in 10 Eppendorfs of 1.5 ml. <br /> | ||
| Line 937: | Line 938: | ||
<p> | <p> | ||
| − | <h6><U> Aim:</U></h6> Check if the PCR works. <br /> | + | <h6><U> Aim :</U></h6> Check if the PCR works. <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 /> | + | <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 /> |
| − | <h6><U>Results:</U></h6><br /><br /> | + | <h6><U>Results :</U></h6><br /><br /> |
<center><img src = "https://static.igem.org/mediawiki/2016/a/ad/Week_10_161Electrophoresis_of_the_PCR_done_on_the_8th_of_August_with_A1-A2-D1-D2.jpg" ; alt "Electrophoresis gel of the PCR"/> | <center><img src = "https://static.igem.org/mediawiki/2016/a/ad/Week_10_161Electrophoresis_of_the_PCR_done_on_the_8th_of_August_with_A1-A2-D1-D2.jpg" ; alt "Electrophoresis gel of the PCR"/> | ||
| − | <i><p> Figure 2: Electrophoresis gel of the PCR</p></i><br /> | + | <i><p> <U>Figure 2 :</U> Electrophoresis gel of the PCR</p></i></center><br /> |
The PCR works properly since we noticed significant bands at the expected level.<br /> | The PCR works properly since we noticed significant bands at the expected level.<br /> | ||
<br /><br /><br /> | <br /><br /><br /> | ||
| Line 957: | Line 958: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> After their ligation in TOPO cloning vector, they will be transformed into TOP 10 cells. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> After their ligation in TOPO cloning vector, they will be transformed into TOP 10 cells. <br /> |
| − | <U>What we did in the lab : </U><br/> | + | <h6><U>Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br /> |
| − | <U>Materials</U>< | + | <h6><U>What we did in the lab :</U></h6><br /> |
| − | • Ligation’s products of A1 | + | <h6><U>Materials</U></h6> |
| − | &bull ; TOP 10 competent cells <br/> | + | • Ligation’s products of A1/A2/D1/D2<br /> |
| − | &bull ; SOC <br/> | + | &bull ; TOP 10 competent cells <br /> |
| − | • Microbiology | + | &bull ; SOC <br /> |
| − | • Xgal at 10 mg | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br /> |
| − | <br/><br/> | + | • Xgal at 10 mg/ml |
| − | <U>Method</U>< | + | <br /><br /> |
| − | 1. Add 6 &# | + | <h6><U>Method</U></h6> |
| − | 2. Put the samples 30 minutes on ice, then 40 seconds at | + | 1. Add 6 µl of ligation product in 50 µl of competent cells.<br /> |
| − | 3. Put the samples 3 minutes on ice.<br/> | + | 2. Put the samples 30 minutes on ice, then 40 seconds at 42°C.<br /> |
| − | 4. Add 150 &# | + | 3. Put the samples 3 minutes on ice.<br /> |
| − | 5. Let incubate 40 minutes at | + | 4. Add 150 µl of SOC.<br /> |
| − | 6. Take LB with carbenicillin petri plate and add Xgal to reach 40 &# | + | 5. Let incubate 40 minutes at 37°C and 150 rpm.<br /> |
| − | 7. Spread bacteria on four distincts petri dishes (one for each insert). | + | 6. Take LB with carbenicillin petri plate and add Xgal to reach 40 µg/ml. As the volume is about 25 ml, add 1 mg of Xgal and as the stock solution is 10 mg/ml, spread 100 µl on the plate.<br /> |
| − | <br/><br/><br/> | + | 7. Spread bacteria on four distincts petri dishes (one for each insert).<br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 988: | Line 990: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> After their ligation we must transform the inserts into bacteria. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> After their ligation we must transform the inserts into bacteria. <br /> |
| − | <U>What we did in the lab : </U><br/> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br /> |
| − | <U>Method</U>< | + | <h6><U>What we did in the lab :</U></h6><br /> |
| − | Add 10 &# | + | <h6><U>Method</U></h6> |
| − | <br/><br/><br/> | + | Add 10 µl of ligation product (to have 100 mg) at the beginning. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,007: | Line 1,010: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Get back the DNA. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> Get back the DNA. <br /> |
| − | <U>What we did in the lab : </U><br/> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br /><br /> |
| − | <U>Method</U>< | + | <h6><U>What we did in the lab : </U><h6><br/> |
| − | We use a final volume of 50 &# | + | <h6><U>Method</U></h6> |
| − | <br/><br/><br/> | + | We use a final volume of 50 µl. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,027: | Line 1,031: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Split the insert and the plasmid. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> Split the insert and the plasmid. <br /> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br /> |
| − | <U>Materials:</U>< | + | <h6><U>What we did in the lab:</U></h6><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/> | + | <h6><U>Materials:</U></h6> |
| − | • Qiagen Miniprep kit <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 /> |
| − | • Digestion enzyme Xba I and Hind III <br/> | + | • Qiagen Miniprep kit <br /> |
| − | • Digestion buffer 2 X <br/> | + | • Digestion enzyme Xba I and Hind III <br /> |
| − | • 1.5 ml Eppendorfs <br/> | + | • Digestion buffer 2 X <br /> |
| − | • Distilled water <br/> | + | • 1.5 ml Eppendorfs <br /> |
| − | • Shaking incubator (INFORS HT)<br/><br/> | + | • Distilled water <br /> |
| − | <U>Method:</U></ | + | • Shaking incubator (INFORS HT)<br /><br /> |
| − | 1. Realize a master mix with : | + | <h6><U>Method:</U></h6> |
| + | 1. Realize a master mix with : <br /><br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table 8</caption> | + | <caption align="bottom" align="center">Table 8 : Volumes</caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,071: | Line 1,076: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | <br/> | + | <br /> |
| − | 2. Put &# | + | 2. Put µl of the master mix in each of the twenty 1.5 ml Eppendorfs and add 5 µl of DNA.<br/> |
| − | 3. Let incubate one hour at | + | 3. Let incubate one hour at 37°C and 150 rpm, then 5 minutes at 65°C<br /><br /> |
| − | <U>Results</U>< | + | <h6><U>Results</U></h6> The 10<sup>th</sup> of August, we realize that we forgot to put Xgal, so we re-spreaded the previous mix on petri dishes with Xgal.<br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,090: | Line 1,095: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> We want to produce 5 &# | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> We want to produce 5 µg of dephosphorylated pET 43.1(a+) from pET 43.1(a+) at 400 ng/ml and we start with the digestion. <br /> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br /> |
| − | <U>Materials:</U>< | + | <h6><U>What we did in the lab :</U></h6><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/> | + | <h6><U>Materials :</U></h6> |
| − | • Qiagen Miniprep kit <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 /> |
| − | • Digestion enzyme Xba I and Hind III <br/> | + | • Qiagen Miniprep kit <br /> |
| − | • CutSmart buffer<br/> | + | • Digestion enzyme Xba I and Hind III <br /> |
| − | • 1.5 ml Eppendorfs <br/> | + | • CutSmart buffer<br /> |
| − | • Distilled water <br/> | + | • 1.5 ml Eppendorfs <br /> |
| − | • Shaking incubator (INFORS HT)<br/><br/> | + | • Distilled water <br /> |
| − | <U>Method:</U></ | + | • Shaking incubator (INFORS HT)<br /><br /> |
| − | 1. Put all the following reactants in a 1.5 ml Eppendorf and let digest one hour at | + | <h6><U>Method :</U></h6> |
| + | 1. Put all the following reactants in a 1.5 ml Eppendorf and let digest one hour at 37°C : <br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table 9</caption> | + | <caption align="bottom" align="center">Table 9 : Volumes</caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,139: | Line 1,145: | ||
</table> | </table> | ||
<br/> | <br/> | ||
| − | 2. Inactivate the enzymes 5 minutes at | + | 2. Inactivate the enzymes 5 minutes at 65°C. <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,155: | Line 1,161: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Get back the digested and purified plasmid before dephosphorylation. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> Get back the digested and purified plasmid before dephosphorylation. <br /><br /> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br /> |
| − | <U>Materials:</U>< | + | <h6><U>What we did in the lab :</U></h6><br /> |
| − | • Products from the digestion of pET 43.1 (a+) with Hind III and | + | <h6><U>Materials :</U></h6> |
| − | • Agarose<br/> | + | • Products from the digestion of pET 43.1(a+) with Hind III and XbaI <br /> |
| − | • Electrophoresis chamber <br/> | + | • Agarose<br /> |
| − | <U>Method:</U></ | + | • Electrophoresis chamber <br /> |
| − | 1. Make a 0.7 | + | <h6><U>Method :</U></h6> |
| − | 2. Prepare the chamber to do the migration at 100 V with two wells for pET43.1(a+) and one for the ladder. <br/> | + | 1. Make a 0.7% agarose gel <br /> |
| − | 3. Take the results and follow the kit steps of Qiagen extraction kit.<br/> | + | 2. Prepare the chamber to do the migration at 100 V with two wells for pET43.1(a+) and one for the ladder. <br /> |
| − | <U>Results | + | 3. Take the results and follow the kit steps of Qiagen extraction kit.<br /><br /> |
| − | We notice two bands for digested pET43.1(a+), one at 6000 bp and another between 1000 and 1500 bp. We extract the bands at 6000 bp.<br/> | + | <h6><U>Results :</U></h6> |
| − | We obtained : <br/> | + | We notice two bands for digested pET43.1(a+), one at 6000 bp and another between 1000 and 1500 bp. We extract the bands at 6000 bp.<br /> |
| − |   m1 = 0.4079 g<br/> | + | We obtained : <br /> |
| − |   m2 = 0 .3720 g<br/> | + |   m1 = 0.4079 g<br /> |
| − | <br/><br/><br/> | + |   m2 = 0 .3720 g<br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,185: | Line 1,192: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Check if the digestion works properly and if we have inserts. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> Check if the digestion works properly and if we have inserts. <br /><br /> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br /> |
| − | <U>Materials:</U>< | + | <h6><U>What we did in the lab:</U></h6><br /> |
| − | • Products from the digestion of C1 <br/> | + | <h6><U>Materials :</U></h6> |
| − | • Agarose<br/> | + | • Products from the digestion of C1 <br /> |
| − | • Electrophoresis chamber <br/> | + | • Agarose<br /> |
| − | <U>Method:</U></ | + | • Electrophoresis chamber <br /> |
| − | 1. Take the 20 &# | + | <h6><U>Method :</U></h6> |
| − | 2. Do the electrophoresis, following the deposit table : <br/> | + | 1. Take the 20 µl of each sample from the digestion and add 4 µl of loading buffer 6X. <br /> |
| − | Ladder | Ø | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | Ø | Ladder <br/> | + | 2. Do the electrophoresis, following the deposit table : <br /><br /> |
| − | Ladder | Ø | 17 | 18 | 19 | 20 <br/> | + | Ladder | Ø | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | Ø | Ladder <br /><br /> |
| − | <U>Results</U>< | + | Ladder | Ø | 17 | 18 | 19 | 20 <br /><br /> |
| − | There is no insert. The colonies do not contain our insert, we must redo the experiment with new colonies. | + | <h6><U>Results</U></h6> |
| − | <br/><br/><br/> | + | There is no insert. The colonies do not contain our insert, we must redo the experiment with new colonies. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,213: | Line 1,221: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Transform the bacterias with our recombined plasmid. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> Transform the bacterias with our recombined plasmid. <br /><br /> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br /> |
| − | <U>Method:</U></ | + | <h6><U>What we did in the lab :</U></h6><br /> |
| − | The volumes of insert are too small, we add 5 &# | + | <h6><U>Method :</U></h6> |
| − | We performed a transformation in DH5α with 1 &# | + | The volumes of insert are too small, we add 5 µl of H<sub>2</sub>O and diluted at 1/100. <br /> |
| − | <br/><br/><br/> | + | We performed a transformation in DH5α with 1 µl of DNA and 50 µl of competent cells. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,233: | Line 1,242: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Have more antibodies. <br/> | + | <p> |
| − | <U>Results</U><br/> We obtained 30 &# | + | <h6><U> Aim :</U></h6> Have more antibodies. <br /><br /> |
| − | <br/><br/><br/> | + | <h6><U>Results :</U></h6><br/> We obtained 30 µ/aliquot. <br /><br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,249: | Line 1,259: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Increase the quantity of colonies containing inserts. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> Increase the quantity of colonies containing inserts. <br /><br /> |
| − | <br/><br/><br/> | + | <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 /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,265: | Line 1,276: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Ligate the insert and the plasmid. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> Ligate the insert and the plasmid. <br /><br /> |
| − | <br/><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 1,282: | Line 1,294: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Transform our inserts in TOP 10 competent cells. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim:</U></h6> Transform our inserts in TOP 10 competent cells. <br /><br /> |
| − | <br/><br/><br/> | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,299: | Line 1,312: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Have bacteria with the right plasmid to produce our protein. <br/> | + | <p> |
| + | <h6><U> Aim:</U> Have bacteria with the right plasmid to produce our protein. <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> 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/> | ||
