Difference between revisions of "Team:Pasteur Paris/Microbiology week11"

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<body>
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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>
 +
 +
 +
    <p><h3><B>August 17, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp1"><h4> Extraction of plasmid DNA </h4></a></br>
 +
        <a href="#exp2"><h4> Digestion of the plasmid pET43.1a(+) with A1 and D1 </h4></a></br>
 +
<a href="#exp3"><h4> Electrophoresis on agarose gel </h4></a></br>
 +
        <a href="#exp4"><h4> Harvest the culture with Midiprep </h4></a></br>
 +
    </p>
 +
    <p><h3><B>August 18, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp5"><h4> Extraction of plasmid DNA </h4></a></br>
 +
        <a href="#exp6"><h4> Purification of the protein </h4></a></br>
 +
        <a href="#exp7"><h4> Protein gel on SDS-Page </h4></a></br>
 +
        <a href="#exp8"><h4> Harvest the culture with Miniprep </h4></a></br>
 +
 +
    </p>
 +
    <p><h3><B>August 19, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp9"><h4> Extraction of plasmid DNA </h4></a></br>
 +
        <a href="#exp10"><h4> Measure the amount of DNA extracted from the miniprep </h4></a></br>
 +
        <a href="#exp11"><h4> Digestion of the plasmid pET43.1a(+) with A1/D1/D2 </h4></a></br>
 +
        <a href="#exp12"><h4> Electrophoresis on agarose gel </h4></a></br>
 +
 +
    </p>
 +
    <p><B><h3>August 21, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp13"><h4> Harvest the culture with Miniprep </h4></a></br>
 +
 +
    </p>
 +
    <p> <h3><B>August 22, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp14"><h4> Extraction of plasmid DNA </h4></a></br>
 +
        <a href="#exp15"><h4> Measure the amount of DNA extracted from the miniprep </h4></a></br>
 +
        <a href="#exp16"><h4> Digestion of the plasmid pET43.1a(+) with A1/D1/D2 </h4></a></br>
 +
        <a href="#exp17"><h4> Electrophoresis on agarose gel </h4></a></br>
 +
        <a href="#exp18"><h4> Harvest the culture with Miniprep gel </h4></a></br>
 +
        <a href="#exp19"><h4> Harvest the culture with Miniprep gel </h4></a></br>
 +
 +
    </p>
 +
 +
 +
    <div class="lightbox" id="exp1">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption>
 +
              <p>
 +
              <U> Aim:</U> To perform a Miniprep to isolate plasmid DNA of pET43.1a(+) with the inserts A1 and D1. The amplification method to increase the amount of plasmid is called Miniprep. </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>Materials:</U></br>
 +
                  &bull; 50 ml Falcon tube</br>
 +
                  &bull; Shaking incubator (INFORS HT)</br>
 +
                  &bull; Swing bucket centrifuge (JOUAN GR41)</br>
 +
                  &bull; QIAGEN Miniprep kit</br>
 +
                  &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
 +
 +
</br></br>
 +
              <U>Method:</U></br>The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.</br>
 +
                      1.Follow QIAGEN kit steps</br>
 +
              </p>
 +
            </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
    <div class="lightbox" id="exp2">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
              <figcaption>
 +
                  <p><U> Aim:</U> To get back our insert from the Miniprep with appropriate enzymes. </br>
 +
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid. </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>Materials:</U></br>
 +
                    &bull; Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB) </br>
 +
                    &bull; Restriction enzyme buffers </br>
 +
                    &bull; 37°C water bath</br>
 +
                    &bull; UV spectrophotometer</br>
 +
                  <U>Method:</U></br>
 +
                      1. Mix all the reagents and let digest during 2 hr at 37°C. </br> Big volumes must be added first!</br>Beginning of digestion 12h10.</br>
 +
                  <table>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Reactants</th>
 +
                            <th>A1</th>
 +
                            <th>D1</th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>DNA</sub></p></strong></td>
 +
                            <td>20 &#181;L </td>
 +
                            <td>20 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>XbaI</sub></p></strong></td>
 +
                            <td>1 &#181;L </td>
 +
                            <td>1 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>HindIII</sub></p></strong></td>
 +
                            <td>1 &#181;L </td>
 +
                            <td>1 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td>
 +
                            <td>5 &#181;L </td>
 +
                            <td>5 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>Buffer Cutsmart (10X)</sub></p></strong></td>
 +
                            <td>3 &#181;L </td>
 +
                            <td>3 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Vol<sub>total</sub></p></strong></td>
 +
                            <td>30 &#181;L </td>
 +
                            <td>30 &#181;L </td>
 +
                      </tbody>
 +
                  </table>
 +
                  <center>Volumes</center></br></br></br>
 +
 +
          2. Incubate 10 min at 65°C to inactivate the enzymes. </br>
 +
          3. Store at -20°C </br>
 +
            </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
    <div class="lightbox" id="exp3">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption>
 +
                <p><U> Aim:</U> This step check the digestion efficiency. Moreover, the inserts will be purified during this step because they will be extracted from the gel.</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>Materials:</U></br>
 +
                          &bull; Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB) </br>
 +
                          &bull; Restriction enzyme buffers </br>
 +
                          &bull; 37°C water bath</br>
 +
                          &bull; UV spectrophotometer</br>
 +
                    <U>Method:</U></br>
 +
                          &bull; Electrophoresis cuve </br>
 +
                          &bull; TAE 1X </br>
 +
                          &bull; Gene ruler (Thermoscientific 1kb plus) </br>
 +
                          &bull; Loading dye </br>
 +
                          &bull; Agarose </br>
 +
                          &bull; UV table </br>
 +
                          &bull; BET </br></br></br>
 +
                    <U>Method:</U></br>Beginning of the electrophoresis at 14h30 at 100V. </br></br></br>
 +
                    <U>Results:</U></br>The gel reveals that A1 contains the insert but the amount of DNA is too low so we will redo the experiment.</br>
 +
                </p>
 +
            </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
 +
<div class="lightbox" id="exp4">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
        <p>
 +
            <U> Aim:</U>  To start a culture for Midiprep. </br>In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. </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>Materials:</U></br>
 +
                &bull; Microbiology equipement </br>
 +
                &bull; 25 ml flasks </br>
 +
                &bull; Carbenicillin 50 mg/ml </br>
 +
                &bull; Chloramphenicol 34 mg/ml </br>
 +
                &bull; LB medium </br></br> </br>
 +
            <U>Method:</U></br>
 +
                  2.One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin at 50 μg/ml. This step is done with the inserts A1/A2/D1/D2 and B1/B2 for sequencing. </br>
 +
                  3.The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br></br> </br>
 +
        </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
<div class="lightbox" id="exp5">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
      <figcaption>
 +
        <p>
 +
            <U> Aim:</U> To perform a Midiprep to isolate plasmid DNA of pET43.1a(+) with A1/A2/D1/D2 and B1/B2 for sequencing.</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>Materials:</U>
 +
</br>
 +
 +
&bull; 50 ml Falcon tube </br>
 +
&bull; Shaking incubator (INFORS HT) </br>
 +
&bull; Swing bucket centrifuge (JOUAN GR41) </br>
 +
&bull; QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)
 +
</br> </br>
 +
 +
<U>Method:</U></br>
 +
The protocol in step 1 ask for spinning at 6000 g but we can only achieve 3500 g so we used 3500 g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below. </br>
 +
 +
1. Use culture from overnight (17 hr) step on June 7, 2016 </br>
 +
2. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500 g, 4°C) </br>
 +
3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet </br>
 +
4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue. </br>
 +
5. Prepare syringes with their cap and the reservoir (on 50 ml Falcon) </br>
 +
6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white. </br>
 +
7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer) </br>
 +
8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer </br>
 +
9. Prepare 10 tubes of 2 ml to aliquot pET43.1a(+) and pSB1C3.
 +
Because we have only bench microfuges, we need to dispense our volume in smaller fractions. </br>
 +
10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes </br>
 +
11. Centrifuge (30 min, 15000 g, room temperature) </br>
 +
12. Add 3.5 ml of isopropanol, mix to precipitate the DNA </br>
 +
13. Centrifuge (30 min, 15 000 g, at RT) </br>
 +
14. Remove isopropanol with pipet without taking DNA and place into chemical waste container </br>
 +
15. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000 g, RT) and let air dry.
 +
16. Resuspend in 50 &#181;L of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C.</br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
<div class="lightbox" id="exp7">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> The previous purification shows a significant band at 30kDa for the samples 22 and 24 but also one at 70kDa. It probably left some NusA in our column so, it will be cleaned.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>Materials:</U>
 +
</br>
 +
 +
&bull;  Fast Purification Liquid Chromatography </br>
 +
&bull;  Chaotropic reagent (Guanidinium 6M) </br>
 +
&bull;  EDTA 0,1M </br>
 +
&bull;  PMSF (100mM) </br>
 +
&bull;  Ni 2+ solution (100mM) </br>
 +
&bull;  Centrifuge (labo deshmukh)
 +
</br></br>
 +
<U>Method:</U></br>
 +
1. Melt the pellet of bacteria C2 (from 1 L culture) and resuspend it with 10 ml of buffer A </br>
 +
2. Put the column off the FPLC and wash it with 20 ml of milliQ water thanks to a fingerpit ans a syringue. </br>
 +
3. Add 20 ml of chaotropic reagent to denaturate the proteins fixed to the column </br>
 +
4. Wash the column with 20 ml of water </br>
 +
5. Add 10 ml of EDTA to clean it from nickel </br>
 +
6. Wash with 20 ml of water </br>
 +
7. Add 5ml of Ni solution to charge the column. The column turns green. </br>
 +
8. Wash with 20 ml of water </br>
 +
9. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication, Finally, the sample is 40 ml, add 40 &microL of PMSF to avoid protein denaturation. </br>
 +
10. Centrifuge 25 min at 16000 g (rotor JA 25.50) </br>
 +
11. Inject your sample in the FPLC </br>
 +
12. Get back several samples: </br>
 +
&bull; C= Crude extract : before centrigugation </br>
 +
&bull; P= Pellet </br>
 +
&bull; SN= Supernatant </br>
 +
&bull; F= Flow through (unfixed proteins) </br>
 +
&bull; W= Wash 5% of buffer B </br>
 +
&bull; Fractions (depending on the gradient) </br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
<div class="lightbox" id="exp7">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p ><U> Aim:</U> Get the size of the protein purified thanks to FPLC in order to know if it is our protein  </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>Materials:</U>
 +
</br>
 +
<U>Materials:</U>
 +
</br>
 +
&bull; SDS-Page cuve </br>
 +
&bull; SDS-Page gel (BIORAD) </br>
 +
&bull; Protein migration buffer </br>
 +
&bull; Protein ladder </br>
 +
&bull; Laemmli 2X </br>
 +
&bull; Coomassie Blue </br>
 +
&bull; Microbiology equipment (Follow this link) </br> </br>
 +
 +
<U>Method:</U></br>
 +
1. In 9 1.5 ml eppendorf, put 20 &#181;L of a sample and 20 &#181;L of Laemmli 2X. </br>
 +
2. Place the gel into the cuve and fill it with migration buffer </br>
 +
3. Follow the next deposit table: </br>
 +
&bull; Protein ruler 8ul </br>
 +
&bull; Pellet </br>
 +
&bull; Supernatant </br>
 +
&bull; Wash </br>
 +
&bull; Flow through </br>
 +
&bull; Fraction 13 </br>
 +
&bull; Fraction 16 </br>
 +
&bull; Fraction 20 </br>
 +
&bull; Fraction 22 </br>
 +
&bull; Fraction 24 </br>
 +
4. Launch the migration at 120V (start at 12h10 and stop at 14h10). </br>
 +
5. Wash the gel three times with distilled water during 5min. </br>
 +
6. Color the gel with Coomassie Blue diluted 1/5 during 30min. </br>
 +
7. Wash with distilled water for 5min then let wash 15min. </br> </br>
 +
 +
<U>Method:</U></br>
 +
We notice a 30kDa band in the well 9 and 10 so we redo a gel with the fractions 21 to 25. Follow exactly the same protocol but with 30 &microL of DNA and 30 &microL of Laemmli 2X. </br>
 +
Deposit table: </br>
 +
- Protein ruler 8 &#181;l </br>
 +
- Fraction 19 </br>
 +
- Fraction 20 </br>
 +
- Fraction 21 </br>
 +
- Fraction 22 </br>
 +
- Fraction 23 </br>
 +
- Fraction 24 </br>
 +
- Fraction 25 </br>
 +
- Fraction 26 </br>
 +
- Fraction 27 </br>
 +
We notice a 30kDa band in the fractions 19 to 21 that may correspond to our protein and a 70kDa band due to NusA in fractions 23 to 25.</br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 
 +
<div class="lightbox" id="exp8">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> To start a culture for Miniprep. </br>
 +
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. </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>Materials:</U>
 +
</br>
 +
<U>Materials:</U>
 +
</br>
 +
&bull; Microbiology equipement </br>
 +
&bull; 15 ml Falcon tube </br>
 +
&bull; Carbenicillin 50 mg/ml </br>
 +
&bull; LB medium </br> </br>
 +
 +
<U>Method:</U></br>
 +
17. One colony is picked from the plates and shaken in 3 ml of LB supplemented with Carbenicillin at 50 &#181;g/ml. This step is done with the inserts A1/A2/D1/D2/C2 and  two colonies B1. </br>
 +
18. The Falcon tube is placed in a shaking incubator at 37°C, 150 rpm overnight. </br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
<div class="lightbox" id="exp9">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> To perform a Miniprep to isolate plasmid DNA of pET43.1a(+) with the inserts A1 (4 tubes) / B1(2 tubes) / C2 (1 tube) / D1 (2 tubes) and D2 (1 tube). </br>
 +
The amplification method to increase the amount of plasmid is called Miniprep. </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>Materials:</U>
 +
</br>
 +
&bull;  Shaking incubator (INFORS HT) </br>
 +
&bull;  Swing bucket centrifuge (JOUAN GR41) </br>
 +
&bull;  QIAGEN Miniprep kit </br>
 +
&bull;  Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) </br> </br>
 +
 +
<U>Method:</U></br>
 +
  The protocol in step 1 ask for spinning at 6000 g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below. </br>
 +
 +
19. Follow QIAGEN kit steps </br> </br>
 +
</p>
 +
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
<div class="lightbox" id="exp10">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)</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>Materials:</U>
 +
</br>
 +
 +
&bull; Nanodrop (Thermofisher)</br>
 +
&bull; Elution buffer from QIAGEN kit</br>
 +
&bull; Microbiology equipment (Follow this link)</br></br>
 +
 +
<U>Method:</U></br>
 +
Analyze absorbance at 260nm</br>
 +
1. Clean the Nanodrop with water</br>
 +
2. Make the blank with 1 &#181;l of elution buffer</br>
 +
3. Put 1ul of your sample on the Nanodrop</br>
 +
4. Make the measure and clean the Nanodrop between each measure</br></br>
 +
 +
<U>Results:</U></br>
 +
<table>
 +
  <thead>
 +
    <tr>
 +
      <th>&#x3BB;= 260 nm</th>
 +
      <th>B1(1)</th>
 +
      <th>B1(2)</th>
 +
      <th>C2>th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td><strong><p>A<sub>DNA</sub></p></strong></td>
 +
<td>0.725</td>
 +
      <td>0.741</td>
 +
      <td>0.761</td>
 +
    </tr>
 +
    <tr>
 +
      <tr>
 +
      <td><strong>C final</strong></td>
 +
      <td>36.3 ng/&#181;l</td>
 +
      <td>37.0 ng/&#181;l</td>
 +
      <td>38.0 ng&#181;l</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<U>Absorbance</U>
 +
</br></br></br>
 +
 +
5. Preparation of the samples for sequencing: put 15 &#181;l of DNA and add 2 &#181;l of fitted primers</br>
 +
6. Send the samples for sequencing</br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp11">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> To get back our insert from the miniprep with appropriate enzymes.</br>
 +
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.</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>Materials:</U>
 +
</br>
 +
 +
&bull; Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB) </br>
 +
&bull; Restriction enzyme buffers </br>
 +
&bull; 37°C water bath </br>
 +
&bull; UV spectrophotometer </br> </br>
 +
 +
<U>Method:</U></br>
 +
4. Mix all the reagents and let digest during 2 hr at 37°C </br>
 +
Big volumes must be added first! </br>
 +
 +
 +
Beginning of digestion 11h45. </br>
 +
 +
<table>
 +
  <thead>
 +
    <tr>
 +
      <th>Reactants</th>
 +
      <th>Each sample</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>DNA</sub></p></strong></td>
 +
      <td>30  &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>XbaI</sub></p></strong></td>
 +
      <td>1  &#181;L</td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>HindIII</sub></p></strong></td>
 +
      <td>1  &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td>
 +
      <td>13  &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>Buffer Cutsmart (10X)</sub></p></strong></td>
 +
      <td>5  &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>total</sub></p></strong></td>
 +
      <td>50  &#181;L </td>
 +
  </tbody>
 +
</table>
 +
Volumes
 +
</br></br></br>
 +
 +
2. Incubate 10 min at 65°C to inactivate the enzymes.</br>
 +
3. Store at -20°C</br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp12">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> This step check the digestion efficiency of A1 (4 tubes)/D1 (2 tubes)/ D2 (1 tube). Moreover, the inserts will be purified during this step because they will be extracted from the gel.</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>Materials:</U>
 +
</br>
 +
 +
&bull; Electrophoresis cuve </br>
 +
&bull; TAE 1X </br>
 +
&bull; Gene ruler (Thermoscientific 1kb plus) </br>
 +
&bull; Loading dye </br>
 +
&bull; Agarose </br>
 +
&bull; UV table </br>
 +
&bull; BET </br>
 +
</br>
 +
 +
<U>Method:</U></br>
 +
Each well will contain 30 &microL of DNA and 6 &microL of Loading Dye. </br>
 +
Follow the next deposit table :</br>
 +
Ladder (6 &microL) / A1 (1) / A1 (2) / A1 (3) / A1 (4) / D1 (1) / D1 (2) / D2 (2)</br>
 +
Beginning of the electrophoresis at 14h20 at 100 V.</br> </br>
 +
 +
<U>Results:</U></br>
 +
The gel reveals that A1 (1) and (2) contains the insert but the amount of DNA is too high to be purified. The other clones do not have an insert.
 +
</br> </br>
 +
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp13">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> To start a culture for Midiprep of insert C2 and B1. </br>
 +
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.</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>Materials:</U>
 +
</br>
 +
&bull; Microbiology equipement </br>
 +
&bull; 25 ml flasks</br>
 +
&bull; Carbenicillin 50 mg/ml</br>
 +
&bull; LB medium</br></br>
 +
 +
<U>Method:</U></br>
 +
5. One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin at 50 &#181;g/ml.</br>
 +
The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp14">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U> To perform a midiprep to isolate plasmid DNA pET43.1a(+) with the inserts C2 and B1.</br>
 +
The amplification method to increase the amount of plasmid is called Mini or Midiprep.</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>Materials:</U>
 +
</br>
 +
&bull; 50 ml Falcon tube</br>
 +
&bull; Shaking incubator (INFORS HT)</br>
 +
&bull; Swing bucket centrifuge (JOUAN GR41)</br>
 +
&bull; QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)</br></br>
 +
 +
<U>Method:</U></br>  The protocol in step 1 ask for spinning at 6000 g but we can only achieve 3500 g so we used 3500 g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below.</br>
 +
 +
6. Use culture from overnight (17 hr) step on June 7, 2016 </br>
 +
7. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500 g, 4°C)</br>
 +
8. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet </br>
 +
9. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue.</br>
 +
10. Prepare syringes with their cap and the reservoir (on 50 ml Falcon)</br>
 +
11. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white.</br>
 +
12. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer)</br>
 +
13. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer</br>
 +
14. Prepare 10 tubes of 2 ml to aliquot pET43.1(+) and pSB1C3. </br>
 +
Because we have only bench microfuges, we need to dispense our volume in smaller fractions.</br>
 +
1. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes</br>
 +
2. Centrifuge (30 min, 15000g, room temperature)</br>
 +
3. Add 3.5 ml of isopropanol, mix to precipitate the DNA</br>
 +
4. Centrifuge (30 min, 15 000g, at RT)</br>
 +
5. Remove isopropanol with pipet without taking DNA and place into chemical waste container</br>
 +
6. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000 g, RT) and let air dry</br>
 +
7. Resuspend in 50 &microL of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C</br></br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp15">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U>  Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)</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>Materials:</U>
 +
</br>
 +
&bull; Nanodrop (Thermofisher)</br>
 +
&bull; Elution buffer from QIAGEN kit</br>
 +
&bull; Microbiology equipment (Follow this link)</br></br>
 +
 +
<U>Method:</U></br>
 +
Analyze absorbance at 260 nm</br>
 +
15. Clean the Nanodrop with water</br>
 +
16. Make the blank with 1ul of elution buffer</br>
 +
17. Put 1ul of your sample on the Nanodrop</br>
 +
18. Make the measure and clean the Nanodrop between each measure</br></br>
 +
 +
<U>Results:</U></br>
 +
<table>
 +
  <thead>
 +
    <tr>
 +
      <th>&#x3BB;= 260 nm</th>
 +
      <th>B1(a)</th>
 +
      <th>B1(b)</th>
 +
      <th>C2(a)</th>
 +
      <th>C2(b)</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td><strong><p>A<sub>260</sub></p></strong></td>
 +
      <td>1.057/td>
 +
      <td>1.323</td>
 +
      <td>0.971</td>
 +
      <td>0.148</td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong>A<sub>280</sub></strong></td>
 +
      <td>0.627</td>
 +
      <td>0.698</td>
 +
      <td>0.571</td>
 +
      <td>0.104</td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong>A<sub>260</sub>/A<sub>280</sub></strong></td>
 +
      <td>1.69</td>
 +
      <td>1.89</td>
 +
      <td>1.70</td>
 +
      <td>1.43</td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong>C final</strong></td>
 +
      <td>52.8 ng/&#181;l</td>
 +
      <td>66.2 ng&#181;l</td>
 +
      <td>48.6 ng/&#181;l</td>
 +
      <td>7.4 ng/&#181;l</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
Absorbance
 +
</br></br></br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
<div class="lightbox" id="exp16">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U>To get back our insert from the miniprep with appropriate enzymes.</br>
 +
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.</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>Materials:</U>
 +
</br>
 +
&bull;  Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB) </br>
 +
&bull;  Restriction enzyme buffers </br>
 +
&bull;  37°C water bath </br>
 +
&bull;  UV spectrophotometer </br> </br>
 +
 +
<U>Method:</U></br>
 +
19. Mix all the reagents and let digest during 2 hr at 37°C </br>
 +
Big volumes must be added first! Make a global mix to be more accurate</br>
 +
</br>
 +
 +
 +
<table>
 +
  <thead>
 +
    <tr>
 +
      <th>Reactants</th>
 +
      <th>Each sample</th>
 +
      <th>Global mix</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>DNA</sub></p></strong></td>
 +
      <td>45 &#181;L </td>
 +
      <td>0 &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>XbaI</sub></p></strong></td>
 +
      <td>2.25 &#181;L </td>
 +
      <td>65.25 &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>HindIII</sub></p></strong></td>
 +
      <td>2.25 &#181;L </td>
 +
      <td>65.25 &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>H<span>2</span>O</sub></p></strong></td>
 +
      <td>1.125 &#181;L</td>
 +
      <td>32.65 &#181;L</td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>Buffer Cutsmart (10X)</sub></p></strong></td>
 +
      <td>5.63 &#181;L </td>
 +
      <td>163.12 &#181;L </td>
 +
    </tr>
 +
    <tr>
 +
      <td><strong><p>Vol<sub>total</sub></p></strong></td>
 +
      <td>56 /&#181;L </td>
 +
      <td>326.27 /&#181;L </td>
 +
  </tbody>
 +
</table>
 +
Volumes
 +
</br></br></br>
 +
 +
2. Incubate 10 min at 65°C to inactivate the enzymes.</br>
 +
3. Store at -20°C
 +
</br></br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
<div class="lightbox" id="exp17">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U>This step check the digestion efficiency of E1(3 tubes) / E2 (12 tubes) / B2(14 tubes). Moreover, the inserts will be purified during this step because they will be extracted from the gel. </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>Materials:</U>
 +
</br>
 +
</br>
 +
&bull; Electrophoresis cuve </br>
 +
&bull; TAE 1X </br>
 +
&bull; Gene ruler (Thermoscientific 1kb plus) </br>
 +
&bull; Loading dye </br>
 +
&bull; Agarose </br>
 +
&bull; UV table </br>
 +
&bull; BET </br>
 +
 +
 +
Method: Each well can contain 40 &#181;l so we made two big gels with 20x2 lines on each. </br> Each sample will contain 62 &#181;l as we add 7 &#181;l of loading dye. They will be divided into two wells. </br>
 +
Deposit table (/// means EMPTY to make the cut easier) </br>
 +
 +
Gel 1 Line 1 : </br>
 +
Ladder /// B2(2) / B2(2) /// B2(3) / B2(3) /// B2(4) / B2(4) /// B2(5) / B2(5) /// B2(1) / B2(1) /// B2(6) / B2(6) </br> </br>
 +
 +
Gel 1 Line 2 : </br>
 +
Ladder /// B2(7) / B2(7) /// B2(8) / B2(8) /// B2(9) / B2(9) /// B2(10) / B2(10) /// B2(11) / B2(11) /// B2(12) / B2(12) </br> </br>
 +
 +
 +
Gel 2 Line 1 : </br>
 +
Ladder /// E1(1) / E1(1) /// E1(2) / E1(2) /// E1(3) / E1(3) /// E2(1) / E2(1) /// E2(2) / E2(2) /// E2(3) / E2(3) /// E2(4) / E2(4) /// E2(5) / E2(5) /// E2(6) / E2(6) /// E2(7) / E2(7) </br> </br>
 +
 +
Gel 2 Line 2 : </br>
 +
Ladder /// E2(8) / E2(8) /// E2(9) / E2(9) /// E2(10) / E2(10) /// E2(11) / E2(11) /// E2(12) / E2(12) /// E2(13) / E2(13) /// B2(13) / B2(13) /// B2(14) / B2(14) /// </br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp18">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U>To start a culture for Miniprep of insert A1, A2, D1 and D2. </br>
 +
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. </br> </br>
 +
 +
</br></br>
 +
<U>What we did in the lab:</U>
 +
</br>
 +
<U>Materials:</U>
 +
</br>
 +
&bull; Microbiology equipement </br>
 +
&bull; 25 ml flasks </br>
 +
&bull; Carbenicillin 50 mg/ml </br>
 +
&bull; LB medium </br> </br>
 +
 +
<U>Method:</U></br>
 +
1. One colony is picked from the plates and shaken in 1.0 ml of LB supplemented with Carbenicillin at 50 μg/ml. 10 colonies are taken from each insert. </br>
 +
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
 +
</br> </br>
 +
Start incubation at 16h30 </br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
<div class="lightbox" id="exp19">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p><U> Aim:</U>To start a culture for Miniprep of insert A1, A2, D1 and D2. </br>
 +
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. </br> </br>
 +
 +
</br></br>
 +
<U>What we did in the lab:</U>
 +
</br>
 +
<U>Materials:</U>
 +
</br>
 +
&bull; Microbiology equipement </br>
 +
&bull; 25 ml flasks </br>
 +
&bull; Carbenicillin 50 mg/ml </br>
 +
&bull; LB medium </br> </br>
 +
 +
<U>Method:</U></br>
 +
1. One colony is picked from the plates and shaken in 1.0 ml of LB supplemented with Carbenicillin at 50 &#181g/ml. 10 colonies are taken from each insert. </br>
 +
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
 +
</br> </br>
 +
Start incubation at 16h30 </br> </br>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
</div>
 +
</div>
 +
 +
</body>
 +
 +
</html>

Revision as of 13:30, 15 October 2016