Difference between revisions of "Team:Pasteur Paris/Microbiology week10"
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| + | <div> | ||
| + | <h2><B>Microbiology Notebook</B></h2> | ||
| + | </div> | ||
| + | |||
| + | <div id="home"> | ||
| + | <center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center> | ||
| + | </div> | ||
<div id="week10"> | <div id="week10"> | ||
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<a href="#exp2"><h4> 151. Digestion of B2, E1 and C2 from the 4<sup>th</sup> of August </h4></a><br/> | <a href="#exp2"><h4> 151. Digestion of B2, E1 and C2 from the 4<sup>th</sup> of August </h4></a><br/> | ||
<a href="#exp3"><h4> 152. Electrophoresis with the results of digestion </h4></a><br/> | <a href="#exp3"><h4> 152. Electrophoresis with the results of digestion </h4></a><br/> | ||
| − | <a href="#exp4"><h4> 153. PCR of inserts A1 | + | <a href="#exp4"><h4> 153. PCR of inserts A1/A2/D1/D2 </h4></a><br/> |
| − | <a href="#exp5"><h4> 154. Gel extraction of A1 | + | <a href="#exp5"><h4> 154. Gel extraction of A1/A2/D1/D2 </h4></a><br/> |
<a href="#exp6"><h4> 155. Gel extraction of B2, E1 and E2 </h4></a><br/> | <a href="#exp6"><h4> 155. Gel extraction of B2, E1 and E2 </h4></a><br/> | ||
| − | <a href="#exp7"><h4> 156. Resuspension of inserts B2 | + | <a href="#exp7"><h4> 156. Resuspension of inserts B2/E1/E2 </h4></a><br/> |
</p> | </p> | ||
<p><h3><B>August 9, 2016:</B></h3></p> | <p><h3><B>August 9, 2016:</B></h3></p> | ||
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<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 301: | ||
• 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 /> | ||
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1. Realize a Mastermix and store it on ice : <br /> | 1. Realize a Mastermix and store it on ice : <br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 113</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 395: | Line 403: | ||
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 | + | <i><p> <U>Figure 18 :</U> Gel of the results of digestion </i></p></center><br /> |
<br /><br /><br /> | <br /><br /><br /> | ||
</p> | </p> | ||
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1. Prepare the following tubes :<br /> | 1. Prepare the following tubes :<br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 114</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
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3. Add 1 µl of DNA following the number of tubes : <br /><br /> | 3. Add 1 µl of DNA following the number of tubes : <br /><br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 115</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
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  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 = | + | <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 | + | <i><p> <U>Figure 19 :</U> Extraction gel of A1-A2-D1-D2 </p></i></center> |
<br /><br /> <br /> | <br /><br /> <br /> | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
| Line 573: | Line 582: | ||
<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> | ||
Follow the Qiagen Extraction gel kit steps with :<br /><br /> | Follow the Qiagen Extraction gel kit steps with :<br /><br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 116</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
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<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 /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 117</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
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Use the following volumes : <br /> | Use the following volumes : <br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 118</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 739: | Line 748: | ||
<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> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 119</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 890: | Line 899: | ||
• 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 919: | ||
<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 927: | ||
• 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 946: | ||
<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 | + | <i><p> <U>Figure 20 :</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 966: | ||
<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> | ||
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<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 1,018: | ||
<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 1,039: | ||
<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 | + | <caption align="bottom" align="center"><i><p> <U>Table 120</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,071: | Line 1,084: | ||
</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 1,103: | ||
<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 | + | <caption align="bottom" align="center"><i><p> <U>Table 121</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,139: | Line 1,153: | ||
</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 1,169: | ||
<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 1,200: | ||
<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 1,229: | ||
<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 1,250: | ||
<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 1,267: | ||
<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 1,284: | ||
<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 1,302: | ||
<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 1,320: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Have bacteria with the right plasmid to produce our protein. <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> Have bacteria with the right plasmid to produce our protein. <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 /> |
| − | • Digested B1 and C2 <br/> | + | <h6><U>Materials:</U></h6> |
| − | • BL21DE3 competent cells<br/> | + | • Digested B1 and C2 <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/> | + | • BL21DE3 competent cells<br /> |
| − | • Shaking incubator (INFORS HT)<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 /> |
| − | • SOC <br/> | + | • Shaking incubator (INFORS HT)<br /> |
| − | <U>Method:</U></ | + | • SOC <br /> |
| − | 1. Take 3 samples of C2 v2 (10, 13 and 16) and 2 samples of B1 v2 (6 and 8).<br/> | + | <h6><U>Method:</U></h6> |
| − | 2. Put 1 &# | + | 1. Take 3 samples of C2 v2 (10, 13 and 16) and 2 samples of B1 v2 (6 and 8).<br /> |
| − | 3. Put the samples 30 minutes on ice and then 40 seconds at | + | 2. Put 1 µl of DNA in 99 µl of H<sub>2</sub>O. Then, put 1 µl of DNA (B1 v2 or C2 v2) in 50 µl of BL21DE3 competent cells.<br /> |
| − | 4. Put the samples 3 minutes on ice. <br/> | + | 3. Put the samples 30 minutes on ice and then 40 seconds at 42°C.<br /> |
| − | 5. Add 150 &# | + | 4. Put the samples 3 minutes on ice. <br /> |
| − | 6. Spread the mix on a petri dish with LB and carbenicillin.<br/> | + | 5. Add 150 µl of SOC and let incubate 30 minutes at 37°C and 150 rpm.<br /> |
| − | 7. Let incubate overnight at | + | 6. Spread the mix on a petri dish with LB and carbenicillin.<br /> |
| − | <br/><br/><br/> | + | 7. Let incubate overnight at 37°C and 150 rpm.<br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,330: | Line 1,352: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Have the concentration of digested pET 43.1 | + | <p> |
| − | <U>Results</U>< | + | <h6><U> Aim:</U></h6> Have the concentration of digested pET 43.1(a+) <br /> |
| − | <br/><br/><br/> | + | <h6><U>Results</U></h6> Measure the concentration of digested pET 43.1(a+) with a Nanodrop. For the first tube, we find a concentration of 6.8 ng/µl in 46 µl. For the second tube, we find a concentration of 8.8 ng/µl in 46 µl. Then, store the samples at -20°C. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,345: | Line 1,368: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Transform C2 v2 and B1 v2 in pET43.1a(+) and DHα . <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 C2 v2 and B1 v2 in pET43.1a(+) and DHα . <br /> |
| − | <U>Results</U>< | + | <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 /> |
| − | For B1 v2 and C2 v2 in pET43.1(a+), transformations were successful.<br/> | + | <h6><U>Results</U></h6> For B2 (4, 7, 9), E1 (1, 2) and E2 (2, 3, 4, 5, 6, 7) we see small colonies so we let and 25 µl of carbenicillin.<br /> |
| − | For A1, A2 and D1, D2 there were too many bacteria growing so we took a colony, diluted it in LB and then spread it on petri dish with LB, Xgal and carbenicillin. | + | For B1 v2 and C2 v2 in pET43.1(a+), transformations were successful.<br /> |
| − | <br/><br/><br/> | + | For A1, A2 and D1, D2 there were too many bacteria growing so we took a colony, diluted it in LB and then spread it on petri dish with LB, Xgal and carbenicillin. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| − | |||
| Line 1,365: | Line 1,388: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> To produce proteins. <br/> | + | <p> |
| − | <U>What we did in the lab:</U><br/> | + | <h6><U> Aim:</U></h6> To produce proteins. <br /> |
| − | <U>Materials:</U>< | + | <h6><U>What we did in the lab:</U></h6><br /> |
| − | • Spectrophotometer Ultrospec 3100<br/> | + | <h6><U>Materials:</U></h6> |
| + | • Spectrophotometer Ultrospec 3100<br /> | ||
• iPTG at 0.5 M | • iPTG at 0.5 M | ||
| − | br/><br/> | + | <br /><br /> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. Make two measurements separated of 30 minutes : | + | 1. Make two measurements separated of 30 minutes : <br /> |
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 122</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 1,385: | Line 1,409: | ||
<th> 6 </th> | <th> 6 </th> | ||
<th> Time of addition of iPTG </th> | <th> Time of addition of iPTG </th> | ||
| − | <th> Concentration (ng&# | + | <th> Concentration (ng/µl)</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 1,458: | Line 1,482: | ||
</table> | </table> | ||
<br/> | <br/> | ||
| − | 2. When the OD<sub>600 nm</sub> reached 0.7 keep the last measurement and centrifuge for 3 minutes at 8000 g. <br/> | + | 2. When the OD<sub>600 nm</sub> reached 0.7 keep the last measurement and centrifuge for 3 minutes at 8000 g. <br /> |
| − | 3. Throw away the supernatant and store at | + | 3. Throw away the supernatant and store at -20°C. <br /> |
| − | 4. Add iPTG to reach 0.3 mM. <br/> | + | 4. Add iPTG to reach 0.3 mM. <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,475: | Line 1,499: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Make the future ligation easier. <br/> | + | <p> |
| − | <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/> | + | <h6><U> Aim :</U></h6> Make the future ligation easier. <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/c0/T--Pasteur_Paris--dephosphorylation_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> |
| − | • rSAP <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 /> |
| − | • CutSmart buffer<br/> | + | • rSAP <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. Start with tube 2 (8.6 ng&# | + | <h6><U>Method :</U></h6> |
| + | 1. Start with tube 2 (8.6 ng/µl in 46 µl) and use the following mix : <br /> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 123</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
<th> </th> | <th> </th> | ||
| − | <th> Volumes ( &# | + | <th> Volumes ( µl) </th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 1,519: | Line 1,544: | ||
</table> | </table> | ||
<br/> | <br/> | ||
| − | 2. Let incubate 30 minutes at | + | 2. Let incubate 30 minutes at 37°C then 5 minutes at 65°C. <br /> |
| − | 3. Do the same for tube 1. | + | 3. Do the same for tube 1. <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,535: | Line 1,560: | ||
<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>< | + | <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 /> |
| − | < | + | <h6><U> What we did in the lab </U></h6><br /> |
| − | <U>Materials:</U>< | + | <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 /> |
| − | • LB medium <br/> | + | • LB medium <br /> |
| − | • Carbenicillin at 50 mg | + | • Carbenicillin at 50 mg/ml <br /> |
| − | • B1 | + | • B1/E1/E2 in TOPO <br /><br /> |
| − | <U>Method</U>< | + | <h6><U>Method :</U></h6> |
| − | We do 31 precultures with a mix with 1 ml of LB and 1 &# | + | We do 31 precultures with a mix with 1 ml of LB and 1 µl of carbenicillin to have :<br /> |
| − |   3 samples of E1 <br/> | + |   3 samples of E1 <br /> |
| − |   14 samples of B2 <br/> | + |   14 samples of B2 <br /> |
| − |   14 samples of E2 | + |   14 samples of E2 <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,563: | Line 1,588: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Send our insert for sequencing as the transformations in BL21DE3. <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/ | + | <h6><U> Aim :</U></h6> Send our insert for sequencing as the transformations in BL21DE3. <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/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,580: | Line 1,605: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Have precultures to redo the cultures in 4 x 1 l of LB as they grow well. <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/ | + | <h6><U> Aim:</U></h6> Have precultures to redo the cultures in 4 x 1 l of LB as they grow well. <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/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,596: | Line 1,621: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Increase the quantity of plasmid for the next ligation. <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/ | + | <h6><U> Aim:</U></h6> Increase the quantity of plasmid for the next ligation. <br /> |
| − | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br /> | |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 1,612: | Line 1,637: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Increase the quantity of 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/ | + | <h6><U> Aim:</U></h6> Increase the quantity of DNA. <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 /> | |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,628: | Line 1,653: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Check if the colonies we took contain the insert. <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> Check if the colonies we took contain the insert. <br /> |
| − | <U> What we did in the lab </U>< | + | <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 /> |
| − | < | + | <h6><U> What we did in the lab </U></h6><br /> |
| − | <U> Materials </U>< | + | <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 /> |
| − | • Digestion enzyme Xba I and Hind III <br/> | + | • Digestion enzyme Xba I and Hind III <br /> |
| − | • Digestion buffer 2.1 <br/> | + | • Digestion buffer 2.1 <br /> |
| − | • 1.5 ml Eppendorfs <br/> | + | • 1.5 ml Eppendorfs <br /> |
| − | • Distilled water <br/> | + | • Distilled water <br /> |
| − | • Shaking incubator (INFORS HT)<br/> | + | • Shaking incubator (INFORS HT)<br /> |
| − | • Inserts B2 | + | • Inserts B2/E1/E2 <br/><br /> |
| − | <U> Method </U>< | + | <h6><U> Method </U></h6> |
| − | 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">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 124</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
<th> </th> | <th> </th> | ||
| − | <th> Volumes (&# | + | <th> Volumes (µl) </th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
| Line 1,677: | Line 1,702: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | <br/> | + | <br /> |
| − | 2. Let incubate one hour at | + | 2. Let incubate one hour at 37°C , then 5 minutes at -20°C. <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,693: | Line 1,718: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Produce the protein in higher quantity. <br/> | + | <p> |
| − | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/a4/T--Pasteur_Paris--Protein_induction_protocol.pdf">link</a><br/><br/> | + | <h6><U> Aim:</U></h6> Produce the protein in higher quantity. <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/a4/T--Pasteur_Paris--Protein_induction_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> |
| − | • iPTG <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 | + | • iPTG <br /> |
| − | <br/><br/> | + | • Carbenicillin at 50 mg/ml |
| − | <U> Method </U>< | + | <br /><br /> |
| − | 1. In a 2 l Erlenmeyer, put 1 l of LB and 1 ml of carbenicillin and incubate at | + | <h6><U> Method </U></h6> |
| − | 2. For each preculture of 25 ml, put it in a 50 ml Falcon, centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 5 ml of clean LB.<br/> | + | 1. In a 2 l Erlenmeyer, put 1 l of LB and 1 ml of carbenicillin and incubate at 37°C and 150 rpm to warm the liquid.<br /> |
| − | 3.Centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 20 ml of clean LB.<br/> | + | 2. For each preculture of 25 ml, put it in a 50 ml Falcon, centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 5 ml of clean LB.<br /> |
| − | 4. In each 2 l erlenmeyer, add 5 ml of our preculture : 4 erlenmeyer for C2 and 4 for B1. <br/> | + | 3.Centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 20 ml of clean LB.<br /> |
| − | 5. Let incubate at 37 °C ann 150 rpm and measure the DO. <br/> | + | 4. In each 2 l erlenmeyer, add 5 ml of our preculture : 4 erlenmeyer for C2 and 4 for B1. <br /> |
| − | 6. Once the DO reaches 0.7, add 1 ml of iPTG.<br/> | + | 5. Let incubate at 37 °C ann 150 rpm and measure the DO. <br /> |
| − | 7. Let incubate for 3 hours. <br/> | + | 6. Once the DO reaches 0.7, add 1 ml of iPTG.<br /> |
| − | 8. Centrifuge the cultures. <br/> | + | 7. Let incubate for 3 hours. <br /> |
| − | 9. Resuspend the pellet in 10 ml of lysis buffer (See protein purification protocol) in a 50 ml Falcon of known weight.<br/> | + | 8. Centrifuge the cultures. <br /> |
| − | 10. Store at | + | 9. Resuspend the pellet in 10 ml of lysis buffer (See protein purification protocol) in a 50 ml Falcon of known weight.<br /> |
| − | <br/><br/><br/> | + | 10. Store at -20°C. <br /> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 1,725: | Line 1,751: | ||
<figcaption> | <figcaption> | ||
| − | <p><U> Aim:</U> Check if the digestion works. <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. <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 /> |
| − | • Agarose <br/> | + | <h6><U> Materials </U></h6> |
| − | • Qiagen kit <br/> | + | • Agarose <br /> |
| − | • Nanodrop <br/> | + | • Qiagen kit <br /> |
| − | • Electrophoresis chamber <br/> | + | • Nanodrop <br /> |
| + | • Electrophoresis chamber <br /> | ||
• Loading buffer 6X | • Loading buffer 6X | ||
| − | <br/><br/> | + | <br /><br /> |
| − | <U> Method </U>< | + | <h6><U> Method </U></h6> |
| − | 1. Add 10 &# | + | 1. Add 10 µl of loading buffer 6X to reach 50 µl for each sample.<br /> |
| − | 2. Do an electrophoresis and extract the bands of the palsmid digested. We obtaines two tubes : <br/> | + | 2. Do an electrophoresis and extract the bands of the palsmid digested. We obtaines two tubes : <br /> |
| − |   Tube1 : m1 = 1.276 − 0.9964 = 131.2 mg <br/> | + |   Tube1 : m1 = 1.276 − 0.9964 = 131.2 mg <br /> |
| − |   Tube2 : m2 = 1.1524 − 0.9994 = 153.0 mg <br/> | + |   Tube2 : m2 = 1.1524 − 0.9994 = 153.0 mg <br /> |
| − | 3. Follow the Qiagen kit steps for a final volum of 50 &# | + | 3. Follow the Qiagen kit steps for a final volum of 50 µl.<br /> |
| − | 4. Measure the concentration with the Nanodrop to see the results.<br/> | + | 4. Measure the concentration with the Nanodrop to see the results.<br /> |
| − | 5. Store at | + | 5. Store at -20°C.<br /><br /> |
| − | <U>Results</U>< | + | <h6><U>Results</U></h6> |
| − | Tube 1 : 20 .2 ng&# | + | Tube 1 : 20 .2 ng/µl<br /> |
| − | Tube 2 : 24.8 ng&# | + | Tube 2 : 24.8 ng/µl <br /> |
| − | <br/><br/><br/> | + | <br /><br /><br /> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
