Difference between revisions of "Team:Ionis Paris/06 06 16"

 
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                                     <h4 class="blog_topHd">
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                                     <h2 class="blog_topHd"> <font color =”#279AD3”>  
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                                     Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid</h4>
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                                     Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid</font></h2>
 
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                                    <h4 class="blog_topHd">Objectives</h4>                       
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                                  <h3><font color =”94FAF1”> Objectives </font></h3>  
 +
                        
 
             <p>To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid.</p>
 
             <p>To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid.</p>
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                                    <h4 class="blog_topHd">Materials</h4>
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  <h3><font color =”94FAF1”> Materials </font></h3>  
 
                                          
 
                                          
 
                                  
 
                                  
 
                               <li><p>Plasmid DNA : pSB1C3-RFP at 200 pg/µL</p>
 
                               <li><p>Plasmid DNA : pSB1C3-RFP at 200 pg/µL</p>
 
                               </li>
 
                               </li>
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                               <li><p>DH5⍺ competent cells <a href="https://2016.igem.org/Team:Ionis_Paris/05_06_16">(See 05/06/16)</a></p>
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                               <li><p>DH5⍺ competent cells (<a href="https://2016.igem.org/Team:Ionis_Paris/05_06_16"><font color="DeepPink"See 05/06/16</font></a>)</p>
 
                               </li>
 
                               </li>
 
                               <li><p>Petri dish LB+Cm: Cm concentration = 25 µg/mL</p>
 
                               <li><p>Petri dish LB+Cm: Cm concentration = 25 µg/mL</p>
 
                               </li>
 
                               </li>
  
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                                            <h4 class="blog_topHd">Protocol</h4>
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                                        <h3><font color =”94FAF1”> Protocol </font></h3>  
  
 
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                           </ol>
 
                           </ol>
  
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                                          <h4 class="blog_topHd">Results</h4>
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                                    <h3><font color =”94FAF1”> Results </font></h3>  
  
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<b>Expected results :</b>
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<p><font color =”46BB0A”> Expected results :</font></p>
 
<p>Some colonies on the petri dishes LB+Cm plated with 1% of transformed bacteria, more with 10%, a lot with 100% and a bacterial lawn with the pellet.<br/>
 
<p>Some colonies on the petri dishes LB+Cm plated with 1% of transformed bacteria, more with 10%, a lot with 100% and a bacterial lawn with the pellet.<br/>
 
A bacterial lawn is expected on the LB petri dishes (+ control).<br/>
 
A bacterial lawn is expected on the LB petri dishes (+ control).<br/>
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All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.</p>
 
All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.</p>
  
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<p><b>Obtained results:</b><br/>
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<p> <font color =”46BB0A”> Obtained results:</font><br/>
 
We obtained expected results.</p>
 
We obtained expected results.</p>
  
  
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                                          <h4 class="blog_topHd">Interpretation</h4>
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                                        <h3><font color =”94FAF1”> Interpretation</font></h3>
 
<p>The transformation worked, as we obtained satisfying results. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. The DH5α cells are therefore competent.</p>
 
<p>The transformation worked, as we obtained satisfying results. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. The DH5α cells are therefore competent.</p>
  
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Latest revision as of 19:55, 19 October 2016

Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid

Objectives

To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid.

Materials

  • Plasmid DNA : pSB1C3-RFP at 200 pg/µL

  • DH5⍺ competent cells ()

  • Petri dish LB+Cm: Cm concentration = 25 µg/mL

    • Protocol

    1. Thaw 1 tube of DH5α competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into 2 transformation tubes on ice.

    2. Add 1 µL (200 pg) of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

    3. Place the mixture on ice for 30 min. Do not mix.

    4. Heat shock at exactly 42°C for 30 s. Do not mix.

    5. Place on ice for 5 min. Do not mix.

    6. Pipette 950 µL of room temperature SOC into the mixture.

    7. Place at 37°C for 60 min at 250 rpm.

    8. Warm selection plates to 25°C.

    9. Mix the cells thoroughly by flicking the tube and inverting.

    10. Spread 100 µL of each dilution (100%,10%,1%) onto a selection plate.

    11. Pellet the remaining cells (except for the control), discard mostly all the supernatant and resuspend the remaining cells in 100 µL of LB. Spread 100 µL onto a selection plate.

    12. Incubate all the plates O/N at 37°C.

    Results

    Expected results :

    Some colonies on the petri dishes LB+Cm plated with 1% of transformed bacteria, more with 10%, a lot with 100% and a bacterial lawn with the pellet.
    A bacterial lawn is expected on the LB petri dishes (+ control).
    No colony is expected on the LB+Cm petri dish plated with no plasmid (- control).
    All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.

    Obtained results:
    We obtained expected results.

    Interpretation

    The transformation worked, as we obtained satisfying results. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. The DH5α cells are therefore competent.

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