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− | < | + | <h2 class="blog_topHd"> <font color =”#279AD3”> |
− | Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid</ | + | Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid</font></h2> |
</div> | </div> | ||
− | + | <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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+ | <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> | ||
− | <li><p>DH5⍺ competent cells <a href="https://2016.igem.org/Team:Ionis_Paris/05_06_16"> | + | <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> | ||
− | + | <h3><font color =”94FAF1”> Protocol </font></h3> | |
<figure class="postImg waves-effect"> | <figure class="postImg waves-effect"> | ||
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</ol> | </ol> | ||
− | + | <h3><font color =”94FAF1”> Results </font></h3> | |
− | < | + | <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> | ||
− | <p>< | + | <p> <font color =”46BB0A”> Obtained results:</font><br/> |
We obtained expected results.</p> | We obtained expected results.</p> | ||
− | + | <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
To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid. Plasmid DNA : pSB1C3-RFP at 200 pg/µL Petri dish LB+Cm: Cm concentration = 25 µg/mL 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. 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. Place the mixture on ice for 30 min. Do not mix. Heat shock at exactly 42°C for 30 s. Do not mix. Place on ice for 5 min. Do not mix. Pipette 950 µL of room temperature SOC into the mixture. Place at 37°C for 60 min at 250 rpm. Warm selection plates to 25°C. Mix the cells thoroughly by flicking the tube and inverting. Spread 100 µL of each dilution (100%,10%,1%) onto a selection plate. 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. Incubate all the plates O/N at 37°C. 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. Obtained results: 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.
Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid
Objectives
Materials
Protocol
Results
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.
We obtained expected results. Interpretation