Difference between revisions of "Team:Ionis Paris/21 07 16"
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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> | ||
| − | < | + | <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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| − | + | <h3><font color =”94FAF1”> Protocol </font></h3> | |
| − | + | <h5><font color =”#3CB5E1”>Experimental conditions achieved : </font></h5> | |
<figure class="postImg waves-effect"> | <figure class="postImg waves-effect"> | ||
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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> | ||
| − | + | <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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| − | + | <h3><font color =”94FAF1”>Results (obtain the 22/07)</font></h3> | |
| − | + | <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> | ||
| − | + | <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> | ||
| − | + | <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> | ||
Revision as of 18:19, 19 October 2016
The objective is to transforme competent DH5⍺ cells with the ligation products BB1, BB2 and BB3 to create a stock of transformed bacteria. 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 We need 20 LB+Cm plates + 11 LB plates 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. Add the 20 µL / 30 µL plasmid DNA to the cell mixture. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex. Place on ice for 30 min. Do not mix. Heat shock at exactly 42°C for 45 s. Do not mix. Place on ice for 5 min. Do not mix. Pipette 250 µL of room temperature SOC into the mixture. Place at 37°C for 1h at 250 rpm. Warm selection plates to 25°C. Mix the cells thoroughly by flicking the tubes and inverting. Spread the corresponding volume onto each plate. Incubate all the plates O/N at 37°C. 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. 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. 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.
Transformation: competent DH5⍺ cells with ligation products BB1, BB2 and BB3
Objectives
Materials
Protocol
Experimental conditions achieved :
Transformations protocol:
Results (obtain the 22/07)
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).
Interpretation
