Team:Grenoble/Biology/Protocols/Transformation 2

From 2012.igem.org

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     <h1>Transformation of <i>E. coli</i> competent cells (heat shock)</h1>
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     <h1>Protocols</h1>
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     <h2>Goal</h2>
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     In this section you will find the description of our experiments in order to be able to reproduce what we did (if you have the will).  
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    Transform <i>E. coli</i> competent cells whith foreign DNA.
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     <h2>Protocol</h2>
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     <h2>Cell culture</h2>
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Following are the instructions from the
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    <ul>
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<a href="http://openwetware.org/wiki/Transforming_chemically_competent_cells">OpenWetWare website</a>.
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Medium">Medium composition</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/LB">LB Agar plates preparation</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Glycerol">Glycerol stock preparation</a></li>
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<ol>
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    </ul>
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     <li>Thaw TSS cells on ice.</li>
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</section>
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    <li>Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).</li>
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     <li>Let sit for 30 minutes on ice.</li>
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     <h2>BioBricks utilisation</h2>
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    <li>Incubate cells for 30 seconds at 42°C.</li>
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    <ul>
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    <li>Incubate cells on ice for 2 min.</li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/BB">BioBricks extraction</a></li>
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    <li>Add 1 mL of LB medium at room temperature.</li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">Chemically competent <i>E. coli</i> cells production</a></li>
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    <li>Incubate for 1 hour at 37°C on shaker.</li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_1">Transformation of <i>E. coli</i> competent cells (TSS method)</a></li>
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     <li>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">Transformation of <i>E. coli</i> competent cells (heat shock)</a></li>
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     <li>Grow overnight at 37°C.</li>
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     </ul>
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</ol>
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</section>
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<section>
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    <h2>Molecular biological techniques</h2>
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    <ul>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_1">PCR using a GoTaq polymerase</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">PCR using a HF Phusion polymerase</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">Gibson Assembly</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">Restriction enzyme digest</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Ligation">Ligation</a></li>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">Miniprep</a></li>
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     </ul>
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</section>
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<section>
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    <h2>Gel migration</h2>
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    <ul>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">Gel electrophoresis</a></li>
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        <li><a href="https://static.igem.org/mediawiki/2012/2/27/Protocol_nucleospin_extract_II.pdf">DNA extraction</a></li>
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     </ul>
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</section>
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<section>
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    <h2>Test techniques</h2>
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    <ul>
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">"AND" gate test (control of pBAD expression by cAMP and arabinose)</a></li>
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    </ul>
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