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

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                                             <h4 class="blog_topHd">Protocol</h4>
 
                                             <h4 class="blog_topHd">Protocol</h4>
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                                <figure class="postImg waves-effect">
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                                    <img src="https://static.igem.org/mediawiki/2016/a/ac/T--Ionis_Paris--Notebook06_06Table1.png" alt="">
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                                </figure>
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                         <ol>  <li><p>Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer.</p>
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                         <ol>  <li><p>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.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.615</p>
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                               <li><p>Add 1 µL (200 pg) of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex.</b></p>
 
                               </li>
 
                               </li>
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                               <li><p>Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube.</p>
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                               <li><p>Place the mixture on ice for 30 min. Do not mix.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Incubate on ice for 30 min. Do not allow cells to warm up over 4°C.</p>
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                               <li><p>Heat shock at exactly 42°C for 30 s. Do not mix.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Spin cells at 4000 rpm for 10 min at 4°C.</p>
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                               <li><p>Place on ice for 5 min. Do not mix.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Discard supernatant and try to drain all remaining media.</p>
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                               <li><p>Pipette 950 µL of room temperature SOC into the mixture.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Gently resuspend on 10 mL cold 0.1M CaCl2</p>
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                               <li><p>Place at 37°C for 60 min at 250 rpm.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Incubate on ice 20 min</p>
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                               <li><p>Warm selection plates to 25°C.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Centrifuge 10 min at 4,000 rpm at 4°C</p>
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                               <li><p>Mix the cells thoroughly by flicking the tube and inverting.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol</p>
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                               <li><p>Spread 100 µL of each dilution (100%,10%,1%) onto a selection plate.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Transfer in 1.5 mL eppendorf (100 µL/tube)</p>
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                               <li><p>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.</p>
 
                               </li>
 
                               </li>
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                               <li><p>Store at -80°C</p>
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                               <li><p>Incubate all the plates O/N at 37°C.</p>
 
                               </li>
 
                               </li>
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 +
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                                          <h4 class="blog_topHd">Results</h4>
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<b>Expected results :</b>
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<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 dish plated with pSB1C3-RFP (+ control).<br/>
 +
No colony is expected on the 2 petri dishes plated with no plasmid (- control).<br/>
 +
All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.</p>
 +
 +
<p><b>Obtained results:</b><br/>
 +
We obtained expected results.</p>
 +
  
 
                           </ol>
 
                           </ol>
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                      <p><b>NB: The competency of the prepared cells will be tested the 23/03.</p></b>
 
  
 
                                     <div role="tabpanel" class="tab-pane fade" id="popular_post">
 
                                     <div role="tabpanel" class="tab-pane fade" id="popular_post">

Revision as of 09:17, 12 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 (See 05/06/16)

  • 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.

      13. 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 dish plated with pSB1C3-RFP (+ control).
      No colony is expected on the 2 petri dishes 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.

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