Team:Grenoble/Biology/Protocols/Transformation 2

From 2012.igem.org

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     <h1>Protocols</h1>
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     <h1>Transformation of <i>E. coli</i> competent cells (heat shock)</h1>
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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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     <h2>Goal</h2>
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    Transform <i>E. coli</i> competent cells whith foreign DNA.
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     <h2>Cell culture</h2>
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     <h2>Protocol</h2>
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    <ul>
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Following are the instructions from the
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Medium">Medium composition</a></li>
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<a href="http://openwetware.org/wiki/Transforming_chemically_competent_cells">OpenWetWare website</a>.
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/LB">LB Agar plates preparation</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Glycerol">Glycerol stock preparation</a></li>
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    </ul>
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<ol>
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     <li>Thaw TSS cells on ice.</li>
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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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     <h2>BioBricks utilisation</h2>
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     <li>Let sit for 30 minutes on ice.</li>
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    <ul>
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    <li>Incubate cells for 30 seconds at 42°C.</li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/BB">BioBricks extraction</a></li>
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    <li>Incubate cells on ice for 2 min.</li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">Chemically competent <i>E. coli</i> cells production</a></li>
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    <li>Add 1 mL of LB medium at room temperature.</li>
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        <li><a href="http://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>Incubate for 1 hour at 37°C on shaker.</li>
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        <li><a href="http://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>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li>
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     </ul>
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     <li>Grow overnight at 37°C.</li>
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</section>
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</ol>
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    <h2>Molecular biological techniques</h2>
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    <ul>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_1">PCR using a GoTaq polymerase</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">PCR using a HF Phusion polymerase</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">Gibson Assembly</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">Restriction enzyme digest</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Ligation">Ligation</a></li>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">Miniprep</a></li>
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     </ul>
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    <h2>Gel migration</h2>
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    <ul>
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        <li><a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">Gel electrophoresis</a></li>
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        <li><a href="http://2012.igem.org/wiki/images/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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    <h2>Test techniques</h2>
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    <ul>
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        <li><a href="http://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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</section>
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Revision as of 05:58, 27 August 2012

iGEM Grenoble 2012

Project

Transformation of E. coli competent cells (heat shock)

Goal

Transform E. coli competent cells whith foreign DNA.

Protocol

Following are the instructions from the OpenWetWare website.

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).
  3. Let sit for 30 minutes on ice.
  4. Incubate cells for 30 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 1 mL of LB medium at room temperature.
  7. Incubate for 1 hour at 37°C on shaker.
  8. Spread 100-300 µL onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37°C.