Difference between revisions of "Team:Pasteur Paris/Test"

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                                         <div data-description="Week_29" class="close_button">
 
                                         <div data-description="Week_29" class="close_button">
 
                                         </div>
 
                                         </div>
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                                        <br>
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                                            </br>
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                                         <br>
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                                         <h2>07.16.2018 </h2></br>
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                                         <p> </p>
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                                         <p>We transformed pET 43.1a and pSB1C3 in DH5-α competent cells, in order to constitute a stock of empty vectors for our manipulation.</p>
−
                                         <br>  
+
                                         <table class="tableData" style="margin: auto;">
 +
                                <tr>
 +
                                    <td><b>Plasmid</b></td>
 +
                                    <td><b>C (ng/μl)</b></td>
 +
                                    <td><b>Volume (μl)</b></td>
 +
                                    <td><b>Competent cell</b></td>
 +
                                    <td><b>Medium</b></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>pET 43.1.a</td>
 +
                                    <td>4.95</td>
 +
                                    <td>1</td>
 +
                                    <td>DH5-α</td>
 +
                                    <td>LB/carbenicilline</td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>pSB1C3</td>
 +
                                    <td>40</td>
 +
                                    <td>1</td>
 +
                                    <td>DH5-α</td>
 +
                                    <td>LB/chloramphenicol</td>
 +
                                </tr>
 +
                                </table>
 +
                                <p>See <a href="https://2018.igem.org/Team:Pasteur_Paris/Protocols/CellBio" style="font-weight:bold;text-decoration:none;color:black;" target="_blank">here</a> the transformation of <i>E. coli </i> DH5-alpha protocol</br>
 +
                                We let the transformed bacteria grow overnight (16 hours).</p> </br></br>
 +
 
 +
 
 +
<h2>07.17.2018</h2></br>
 +
<p><b>Results: </b></br>We went to see our bacterial culture: <ul style="text-align: left; list-style: disc;font-size: 16px;" >
 +
<li>Bacteria transformed with pET 43.1 had not grown.</li>
 +
<li>Bacteria transformed with pSB1C3 had formed colonies.</li></ul></br></p>
 +
<p><b>Interpretations: </b></br>
 +
Bacteria transformed with pET 43.1 did not grow. We found a non-commercial tube of pET 43.1  in the freezer from last year team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. Transformation did not work as expected, probably because:<ul style="text-align: left; list-style: disc;font-size: 16px;" >
 +
<li>There was no DNA left in the tube.</li>
 +
<li>The concentration of DNA was too low .</li></ul></p>
 +
<p></br>We cultivated the transformed pSB1C3 bacteria in liquid medium 2 x 25ml + Chloramphenicol (25µg/ml) overnight at 37°C, 180 rpm.</br></p>
 +
<p><b>Results: </b></br>
 +
Bacteria successfully transformed with pSB1C3.</br>
 +
(See <a href="https://2018.igem.org/Team:Pasteur_Paris/Protocols/CellBio" style="font-weight: bold ; color:black; text-decoration:none;" target="_blank">here</a> the liquid culture protocol</a>)
 +
</br></br>
 +
 
 +
<h2>07.18.2018</h2></br>
 +
<p><b>Extraction</b>:</br>
 +
We extracted the pSB1C3 plasmid from the bacterial culture.</br>
 +
The protocol used was the Qiagen Plasmid Purification Kit (See protocol <a href="https://2018.igem.org/Team:Pasteur_Paris/Protocols/CellBio" style="font-weight: bold ; color:black; text-decoration:none;" target="_blank">here </a>)</br>
 +
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1)
 +
(See the NanoDrop protocol <a href="https://2018.igem.org/Team:Pasteur_Paris/Protocols/CellBio" style="font-weight: bold ; color:black; text-decoration:none;" target="_blank">here</a>)</br></p>
 +
 
 +
<p><b>Results</b>:</br>
 +
We used the Nanodrop to quantify the purified DNA.</br></p>
 +
<table class="tableData" style="margin: auto;">
 +
                                <tr>
 +
                                    <td><b>Sample</b></td>
 +
                                    <td><b>1</b></td>
 +
                                    <td><b>2</b></td>
 +
                                    <td><b>3</b></td>
 +
                                    <td><b>4</b></td>
 +
                                    <td><b>5</b></td>
 +
                                    <td><b>6</b></td>
 +
                                    <td><b>7</b></td>
 +
                                    <td><b>8</b></td>
 +
                                    <td><b>9</b></td>
 +
                                    <td><b>10</b></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Volume (µl)</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                    <td>50</td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>C (ng/µl)</td>
 +
                                    <td>-7.8</td>
 +
                                    <td>-8.5</td>
 +
                                    <td>54.8</td>
 +
                                    <td>5.6</td>
 +
                                    <td>237.4</td>
 +
                                    <td>-7.9</td>
 +
                                    <td>-7.2</td>
 +
                                    <td>-7.5</td>
 +
                                    <td>-6.9</td>
 +
                                    <td>-6.5</td>
 +
                                </tr>
 +
<tr>
 +
                                    <td>260/280</td>
 +
                                    <td>1.43</td>
 +
                                    <td>1.42</td>
 +
                                    <td>1.97</td>
 +
                                    <td>3.04</td>
 +
                                    <td>1.85</td>
 +
                                    <td>1.32</td>
 +
                                    <td>1.47</td>
 +
                                    <td>1.56</td>
 +
                                    <td>1.35</td>
 +
                                    <td>1.44</td>
 +
                                </tr>
 +
                                </table></br>
 +
<p><b>Remarque:</b></br>
 +
[NA] < 0 = no DNA</br>
 +
[NA] > 200 good</br>
 +
        260/280 [1;2]</br></p>
 +
</br>
 +
</br>
 +
 
 +
 
 +
 
 +
 
 +
 
                                     </div>
 
                                     </div>
 +
 +
 +
 +
 +
  
 
                                     <div data-description="Week_29" class="panel" id="pan_2901" style="text-align:left;">
 
                                     <div data-description="Week_29" class="panel" id="pan_2901" style="text-align:left;">

Revision as of 16:18, 14 October 2018

""

Week 29 16 - 22 July
Week 30 23 - 29 July
Week 31 30 July - 5 Aug
Week 32 6 - 12 Aug
Week 33 13 - 19 Aug
Week 34 20 - 26 Aug
Week 35 27 Aug - 2 Sept
Week 36 3 - 9 Sept
Week 37 10 - 16 Sept
Week 38 17 - 23 Sept
Week 39 24 - 30 Sept
Week 40 1 - 7 Oct
Week 41 8 - 14 Oct
Week 42 14 - 17 Oct

Bacteriology

Cell culture

Microfluidics/Membrane

Product Design


07.16.2018


We transformed pET 43.1a and pSB1C3 in DH5-α competent cells, in order to constitute a stock of empty vectors for our manipulation.

Plasmid C (ng/μl) Volume (μl) Competent cell Medium
pET 43.1.a 4.95 1 DH5-α LB/carbenicilline
pSB1C3 40 1 DH5-α LB/chloramphenicol

See here the transformation of E. coli DH5-alpha protocol
We let the transformed bacteria grow overnight (16 hours).



07.17.2018


Results:
We went to see our bacterial culture:

  • Bacteria transformed with pET 43.1 had not grown.
  • Bacteria transformed with pSB1C3 had formed colonies.

Interpretations:
Bacteria transformed with pET 43.1 did not grow. We found a non-commercial tube of pET 43.1 in the freezer from last year team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. Transformation did not work as expected, probably because:

  • There was no DNA left in the tube.
  • The concentration of DNA was too low .


We cultivated the transformed pSB1C3 bacteria in liquid medium 2 x 25ml + Chloramphenicol (25µg/ml) overnight at 37°C, 180 rpm.

Results:
Bacteria successfully transformed with pSB1C3.
(See here the liquid culture protocol)

07.18.2018


Extraction:
We extracted the pSB1C3 plasmid from the bacterial culture.
The protocol used was the Qiagen Plasmid Purification Kit (See protocol here )
Measure of the DNA concentration in each tube thanks to the NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)

Results:
We used the Nanodrop to quantify the purified DNA.

Sample 1 2 3 4 5 6 7 8 9 10
Volume (µl) 50 50 50 50 50 50 50 50 50 50
C (ng/µl) -7.8 -8.5 54.8 5.6 237.4 -7.9 -7.2 -7.5 -6.9 -6.5
260/280 1.43 1.42 1.97 3.04 1.85 1.32 1.47 1.56 1.35 1.44

Remarque:
[NA] < 0 = no DNA
[NA] > 200 good
260/280 [1;2]





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Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Salut




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Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



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Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day


Bacteriology

Cell culture

Microfluidics/Membrane

Product Design



Something was done one that day




Something was done one that day




Something was done one that day




Something was done one that day