Difference between revisions of "Team:Pasteur Paris/Microbiology week6"
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3. put them 1 hour at 37°C and 180 RPM</br> | 3. put them 1 hour at 37°C and 180 RPM</br> | ||
4. Store the master plate (reference) at -4°C and make a copy plate grown at 37°C, then stored at 4°C.</br> | 4. Store the master plate (reference) at -4°C and make a copy plate grown at 37°C, then stored at 4°C.</br> | ||
| − | 5. Measure absorbance at | + | 5. Measure absorbance at 600 nm to make a growth curve (ultrospec 3100 pro, GE Lifesciences) using plain plastic cuvettes, 1 cm path length. |
</br></br> | </br></br> | ||
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<tr> | <tr> | ||
<th>Time</th> | <th>Time</th> | ||
| − | <th>C2 1.1 | + | <th>C2 1.1 Abs 600 nm</th> |
| − | <th>C2 1.2 | + | <th>C2 1.2 Abs 600 nm</th> |
</tr> | </tr> | ||
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2. We let the induction proceed during 3 hours at 37°C and 180 RPM | 2. We let the induction proceed during 3 hours at 37°C and 180 RPM | ||
| − | Measure of absorbance: | + | Measure of absorbance: OD<sub>600 nm</sub> C2 1.1= 0.865, OD<sub>600 nm</sub> C2 1.2 =0.846</br></br> |
<U>Results: </U>Our experiment failed because the agitation was stopped ! (Someone unfortunately either didn't restart the incubator shaking mode or programmed a small time on the shaker's timer by error). We pelleted 1 ml of each culture (3 min at 6800 g) and stored at -20°C. The Erlenmeyer content was pelleted too (5 min at 4500 RPM) and stored at -80°C. </U></br> | <U>Results: </U>Our experiment failed because the agitation was stopped ! (Someone unfortunately either didn't restart the incubator shaking mode or programmed a small time on the shaker's timer by error). We pelleted 1 ml of each culture (3 min at 6800 g) and stored at -20°C. The Erlenmeyer content was pelleted too (5 min at 4500 RPM) and stored at -80°C. </U></br> | ||
In the meanwhile, during the growth curve experiment, we performed all the steps for the transformation of the new versions of our inserts synthesized bu iDT.</U></br> | In the meanwhile, during the growth curve experiment, we performed all the steps for the transformation of the new versions of our inserts synthesized bu iDT.</U></br> | ||
| − | We indeed decided to | + | We indeed decided to re-synthesized our inserts, such that it had a few improvements, namely that now it'll depend on the vector's promoter, the linker regions had less GC content, and were shortened. We will refer to these constructs as X.V2. |
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>after the digestion we dephosphorylate It to prevent that pET43.1 | + | <U> Aim:</U>after the digestion we dephosphorylate It to prevent that pET43.1 to religates without the insert.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <U> Aim:</U>Integrate the inserts into the plasmid</br></br> |
<U> Protocol:</U> follow of July 7, 2016 | <U> Protocol:</U> follow of July 7, 2016 | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Purify the | + | <U> Aim:</U>Purify the digested inserts (C1/C2/B1/B2) and the plasmid before the ligation.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">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> | ||
| − | 1. make | + | 1. make a 0.7% agarose gel</br> |
2. do an electrophoresis following the deposit table</br></br> | 2. do an electrophoresis following the deposit table</br></br> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <U> Aim:</U>To Prepare our plasmids for the transformation.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer | + | <td><strong><p>Buffer 10X</p></strong></td> |
<td>2 µl</td> | <td>2 µl</td> | ||
<td>2 µl</td> | <td>2 µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td><strong><p>H<sub>2</sub>0</p></strong></td> |
<td>9 µl</td> | <td>9 µl</td> | ||
<td>1 µl</td> | <td>1 µl</td> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <U> Aim:</U>Transform our plasmids into competent cells to amplify the DNA. </br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">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> | ||
| − | Moreover, we spread the competent cells from July 7,2016 on petri | + | Moreover, we spread the competent cells from July 7,2016 on petri dishes to have new colonies.</br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> As our culture of BL21DE3 | + | <U> Aim:</U> As our culture of BL21DE3 didn’t work we redid exactly the same experiment as yesterday.</br></br> |
<table> | <table> | ||
<thead> | <thead> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer | + | <td><strong><p>Buffer 10X</p></strong></td> |
<td>2 µl</td> | <td>2 µl</td> | ||
<td>2 µl</td> | <td>2 µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td><strong><p>H<sub>2<sub>0</p></strong></td> |
<td>9 µl</td> | <td>9 µl</td> | ||
<td>1 µl</td> | <td>1 µl</td> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Check | + | <U> Aim:</U>Check if the bacteria that were induced by IPTG have produced the protein (weight = 30 kDa).</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">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> | ||
| − | • mini protean TGX 4-15% precast gels ( | + | • mini protean TGX 4-15% precast gels (BioRad)</br> |
| − | • | + | • TGS Buffer 10X</br> |
| − | • | + | • Electrophoresis chamber</br> |
• Samples of our culture </br></br> | • Samples of our culture </br></br> | ||
C2 1.1 (-) IPTG</br> | C2 1.1 (-) IPTG</br> | ||
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<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | 1. Put the gel in the | + | 1. Put the gel in the electrophoresis chamber, take off the green parts off the gel before</br> |
| − | 2. Fill the | + | 2. Fill the chamber with the buffer 1X</br> |
3. Follow the deposit table:</br> | 3. Follow the deposit table:</br> | ||
| − | L1: 8 µl ladder protein Thermofisher</br> | + | L1: 8 µl ladder protein Thermofisher (PAGE Ruler plus)</br> |
L3: 17 µl of C2 1.1 (-) IPTG</br> | L3: 17 µl of C2 1.1 (-) IPTG</br> | ||
L6: 17 µl of C2 1.1 (+) IPTG</br> | L6: 17 µl of C2 1.1 (+) IPTG</br> | ||
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- 10 µl of protein solution</br> | - 10 µl of protein solution</br> | ||
- 10 µl of laemli 2X</br> | - 10 µl of laemli 2X</br> | ||
| − | Heat the samples at 95°C for 5 min to | + | Heat the samples at 95°C for 5 min to denature the protein</br> |
4. start of migration at 130 V</br> | 4. start of migration at 130 V</br> | ||
5. Stop the migration about one hour later</br> | 5. Stop the migration about one hour later</br> | ||
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<U> Results:</U></br> | <U> Results:</U></br> | ||
| − | It seems to have a slight band for C2 11 (+) IPTG that could be our protein but this band doesn’t appear in C2 1.2 (+) IPTG. We decided to let it the whole weekend in distilled | + | It seems to have a slight band for C2 11 (+) IPTG that could be our protein but this band doesn’t appear in C2 1.2 (+) IPTG. We decided to let it the whole weekend in distilled H<sub>2</sub>0 to analyze the bands.</br></br> |
</p> | </p> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>get back the plasmid | + | <U> Aim:</U>To get back the plasmid from the cultures made on the July 11, 2016.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">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> | ||
| − | Once, the DNA | + | Once, the DNA has been pelleted let the Eppendorf open to evacuate the ethanol (all the week-end).</br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
