Difference between revisions of "Team:Ionis Paris/21 07 16"

 
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                                    <h4 class="blog_topHd">
 
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                                    Transformation: competent DH5⍺ cells with ligation products BB1, BB2 and BB3</h4>
 
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                                     <h4 class="blog_topHd">Objectives</h4>                      
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                                     <h2 class="blog_topHd"> <font color =”#279AD3”>Transformation: competent DH5⍺ cells with ligation products BB1, BB2 and BB3</font></h2>
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                                  <h3><font color =”94FAF1”> Objectives </font></h3>                    
 
             <p>The objective is to transforme competent DH5⍺ cells with the ligation products BB1, BB2 and BB3 to create a stock of transformed bacteria.</p>
 
             <p>The objective is to transforme competent DH5⍺ cells with the ligation products BB1, BB2 and BB3 to create a stock of transformed bacteria.</p>
  
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                                     <h4 class="blog_topHd">Materials</h4>
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                                     <h3><font color =”94FAF1”> Materials </font></h3>
 
    
 
    
 
                               <li><p>6 aliquots of NEB DH5⍺ Competent E.coli (C2287)</p></li>
 
                               <li><p>6 aliquots of NEB DH5⍺ Competent E.coli (C2287)</p></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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                                  <h4>Experimental conditions achieved : </h4>
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                          <h5><font color =”#3CB5E1”>Experimental conditions achieved : </font></h5>  
  
 
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                           <p>We need 20 LB+Cm plates + 11 LB plates</p>
 
                           <p>We need 20 LB+Cm plates + 11 LB plates</p>
 
                                        
 
                                        
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                                      <h4>Transformations protocol:</h4>
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                              <h5><font color =”#3CB5E1”>Transformations protocol:</font></h5>  
  
 
                                             <ol> <li><p>haw 2 tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.        </p></li>
 
                                             <ol> <li><p>haw 2 tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.        </p></li>
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                                          <h4 class="blog_topHd">Results (obtain the 22/07)</h4>
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                                        <h3><font color =”94FAF1”>Results (obtain the 22/07)</font></h3>
  
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                              <h4>Expected results:</h4>
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                        <p><font color= ”46BB0A”> Expected results:</font></p>
  
 
                           <p>Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.<br/>
 
                           <p>Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.<br/>
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No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).</p>
 
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).</p>
  
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                              <h4>Obtained results:</h4>
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                            <p> <font color= ”46BB0A”> Obtained results:</font><br/>
  
 
                           <p>We obtained expected results for petri-dishes plated with bacteria transformed with BB1 and BB2, but there is no colonies for petri-dishes plated with bacteria transformed with BB3.</p>
 
                           <p>We obtained expected results for petri-dishes plated with bacteria transformed with BB1 and BB2, but there is no colonies for petri-dishes plated with bacteria transformed with BB3.</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 for BB1 and BB2. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmids BB1 and BB2. A new digestion, ligation and transformation is necessary for BB3.</p>
 
                         <p>The transformation worked for BB1 and BB2. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmids BB1 and BB2. A new digestion, ligation and transformation is necessary for BB3.</p>
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Latest revision as of 20:06, 19 October 2016

Transformation: competent DH5⍺ cells with ligation products BB1, BB2 and BB3

Objectives

The objective is to transforme competent DH5⍺ cells with the ligation products BB1, BB2 and BB3 to create a stock of transformed bacteria.

Materials

  • 6 aliquots of NEB DH5⍺ Competent E.coli (C2287)

  • Plasmid DNA : Ligation product BB1, BB2 and BB3 (from the 20/07/16)

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

    • Protocol

    Experimental conditions achieved :

    We need 20 LB+Cm plates + 11 LB plates

    Transformations protocol:

    1. haw 2 tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.

    2. Add the 20 µL / 30 µL plasmid DNA to the cell mixture.

    3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.

    4. Place on ice for 30 min. Do not mix.

    5. Heat shock at exactly 42°C for 45 s. Do not mix.

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

    7. Pipette 250 µL of room temperature SOC into the mixture.

    8. Place at 37°C for 1h at 250 rpm.

    9. Warm selection plates to 25°C.

    10. Mix the cells thoroughly by flicking the tubes and inverting.

    11. Spread the corresponding volume onto each plate.

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

    Results (obtain the 22/07)

    Expected results:

    Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
    A bacterial lawn on the LB petri dish without antibiotic.
    No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

    Obtained results:

    We obtained expected results for petri-dishes plated with bacteria transformed with BB1 and BB2, but there is no colonies for petri-dishes plated with bacteria transformed with BB3.

    Interpretation

    The transformation worked for BB1 and BB2. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmids BB1 and BB2. A new digestion, ligation and transformation is necessary for BB3.

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