Difference between revisions of "Team:Pasteur Paris/Week 1"

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               <p style="text-align: justify; padding: 30px;">
 
               <p style="text-align: justify; padding: 30px;">
  
−
<center> <b><u>06-04-15 : Transformation of our biobricks in DH5-α competent cells : xxxxxxxx </b></u></center>
+
<p>1/ Resuspension of DNA in 15µL of DNAse, RNAse free water: BBa_K808007,  BBa_K808010, BBa_K808011, BBa_K808012, BBa_K808013, BBa_K808014, BBa_K936000,  BBa_K316003.<br />
−
<br>
+
  </p>
−
<ul style="text-align: left"><b><i> Materials : </b></i>
+
<p>2/ Storage at -20°C.</p>
−
  <li>Competent DH5α cells (100µL per transformation)</li>
+
<p>3/ Transformation in DH5α Competent cells purchased from Invitrogen (# 18265-017) was done following the transformation  protocol. (BBa_K808007, BBa_K808010, BBa_K808011,  BBa_K808012, BBa_K808013, BBa_K808014, BBa_K936000, BBa_K316003).</p>
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  <li>Ice (in ice bucket/container)</li>
+
<p>4/ Cells were plated on LB + chloramphenicol 34µg/mL (Cm34). </p>
−
  <li>2mL tube (1 per a transformation)</li>
+
<p>5/ Incubation  overnight. </p>
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  <li>42°C water bath</li>
+
<p>6/ Storage at 4°C. </p>
−
  <li>SOC media</li>
+
<p>7/ Plasmid  purification following the MiniPrep protocol. </p>
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  <li>Petri dishes with LB agar and appropriate antibiotic (2 per transformation)</li>
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<div align="center">
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   <li>37°C incubator</li>
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  <table border="1" cellspacing="0" cellpadding="0" width="620">
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</ul>
+
    <tr>
 +
      <td width="100" rowspan="2" valign="top"><p><strong>BBa</strong></p></td>
 +
      <td width="520" colspan="3" valign="top"><p align="center"><strong>Concentration    (ng/µL)</strong></p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="173" valign="top"><p><strong>Clone 1</strong></p></td>
 +
      <td width="173" valign="top"><p><strong>Clone 2</strong></p></td>
 +
      <td width="173" valign="top"><p><strong>Clone 3</strong></p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808007</strong></p></td>
 +
      <td width="173" valign="top"><p>73.4</p></td>
 +
      <td width="173" valign="top"><p>44.3</p></td>
 +
      <td width="173" valign="top"><p>56.3</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808010</strong></p></td>
 +
      <td width="173" valign="top"><p>40.8</p></td>
 +
      <td width="173" valign="top"><p>27.5</p></td>
 +
      <td width="173" valign="top"><p>29.5</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808011</strong></p></td>
 +
      <td width="173" valign="top"><p>28.8</p></td>
 +
      <td width="173" valign="top"><p>30.4</p></td>
 +
      <td width="173" valign="top"><p>18.5</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808012</strong></p></td>
 +
      <td width="173" valign="top"><p>49.5</p></td>
 +
      <td width="173" valign="top"><p>#VALEUR!</p></td>
 +
      <td width="173" valign="top"><p>56.5</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808013</strong></p></td>
 +
      <td width="173" valign="top"><p>18.8</p></td>
 +
      <td width="173" valign="top"><p>26</p></td>
 +
      <td width="173" valign="top"><p>15.7</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>808014</strong></p></td>
 +
      <td width="173" valign="top"><p>30</p></td>
 +
      <td width="173" valign="top"><p>41.4</p></td>
 +
      <td width="173" valign="top"><p>37.4</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>316003</strong></p></td>
 +
      <td width="173" valign="top"><p>51.5</p></td>
 +
      <td width="173" valign="top"><p>23.5</p></td>
 +
      <td width="173" valign="top"><p>36.1</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="100" valign="top"><p><strong>936000</strong></p></td>
 +
      <td width="173" valign="top"><p>15.2</p></td>
 +
      <td width="173" valign="top"><p>17.7</p></td>
 +
      <td width="173" valign="top"><p>13.8</p></td>
 +
    </tr>
 +
   </table>
 +
</div>
 +
<p>&nbsp;</p>
  
−
<br>
 
  
−
<ol style="text-align: left"><b><i>Protocol : </b></i>
 
−
  <li>Thaw on ice one tube of DH5α cells. Place 1.5mL microcentrifuge tubes on wet ice.
 
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  <li>Gently mix cells with the pipette tip and aliquot 50µL for each transformation into a 1.5mL centrifuge tube.
 
−
  <li>Refreeze any unused cells.
 
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  <li>Add 1 to 5µL (1-10ng) DNA to the cell and mix gently (Not mix by pipetting up and down).
 
−
  <li>Incubate tubes on ice for 30 minutes.
 
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  <li>Heat shock cells for 40 seconds in a 42°C water bath without shaking.
 
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  <li>Place tube on ice for 3 minutes.
 
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  <li>Add pre-warmed 700µL SOC medium (XXXXXXXX).
 
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  <li>Incubate at 37°C for 40 minutes under shaking condition (200 rpm).
 
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  <li>Spread 200µL of transformed cell on LB + Cm34 (chloramphenicol 34µg/mL).
 
−
  <li>Incubate dishes overnight at 37°C.
 
−
</ol>
 
  
−
<br>
 
−
<br>
 
  
−
<center><b><u>06-05-15 : Bacterial amplification : xxxxxxxxxxx</b></u></center>
 
  
−
<ul style="text-align: left"><b><i>Materials :</b></i>
 
−
  <li>Petri dishes</li>
 
−
  <li>LB with or without chloramphenicol</li>
 
−
  <li>15mL falcon tubes (3 per Petri dishes used)</li>
 
−
</ul>
 
−
 
−
<br>
 
−
 
−
<ol style="text-align: left"><b><i>Protocol :</b></i>
 
−
  <li>Fill the tubes with 5mL of the correct media (3 tubes per strain).</li>
 
−
  <li>Pick three transformed clones per Petri dishes (1 clone per 15mL tube).</li>
 
−
  <li>Incubate in agitation (8 hours at 200 rpm, 37°C).</li>
 
−
</ol>
 
  
  

Revision as of 21:52, 12 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 1

06/01 - 06/05

1/ Resuspension of DNA in 15µL of DNAse, RNAse free water: BBa_K808007, BBa_K808010, BBa_K808011, BBa_K808012, BBa_K808013, BBa_K808014, BBa_K936000, BBa_K316003.

2/ Storage at -20°C.

3/ Transformation in DH5α Competent cells purchased from Invitrogen (# 18265-017) was done following the transformation protocol. (BBa_K808007, BBa_K808010, BBa_K808011, BBa_K808012, BBa_K808013, BBa_K808014, BBa_K936000, BBa_K316003).

4/ Cells were plated on LB + chloramphenicol 34µg/mL (Cm34).

5/ Incubation overnight.

6/ Storage at 4°C.

7/ Plasmid purification following the MiniPrep protocol.

BBa

Concentration (ng/µL)

Clone 1

Clone 2

Clone 3

808007

73.4

44.3

56.3

808010

40.8

27.5

29.5

808011

28.8

30.4

18.5

808012

49.5

#VALEUR!

56.5

808013

18.8

26

15.7

808014

30

41.4

37.4

316003

51.5

23.5

36.1

936000

15.2

17.7

13.8

 



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