Team:Grenoble/Biology/Protocols/Transformation 2

From 2012.igem.org

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<div id="cadre">
<div id="cadre">
<section>
<section>
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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>
 +
    Transform <i>E. coli</i> competent cells with foreign DNA.
</section>
</section>
<br/>
<br/>
<section>
<section>
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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="https://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" target="blank">OpenWetWare website</a>.
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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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<br/>
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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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<br/>
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    </ul>
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<ol>
-
</section>
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    <li>Prepare ice containers.</li>
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<section>
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    <br/>
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    <h2>BioBricks utilisation</h2>
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    <center>
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    <ul>
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         <table class="tableau">
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/BB">BioBricks extraction</a></li>
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                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
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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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                <tr><td><div class="petit">To prevent the ice container from falling off the workbench, it is
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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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                recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack.  
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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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It is important to know where manipulations will be achieved in order to apprehend the possible risks.</div></td></tr>
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     </ul>
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        </table>
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</section>
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    </center>
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<section>
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    <br/>
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     <h2>Molecular biological techniques</h2>
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    <li>Thaw <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">TSS <i>E. coli</i> competent cells</a> (from the freezer) on ice.</li>
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     <ul>
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    <br/>
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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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    <center>
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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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         <table class="tableau">
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         <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">Gibson Assembly</a></li>
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                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
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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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                <tr><td><div class="petit">Using the -80°C freezer requires to wear special gloves
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Ligation">Ligation</a></li>
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                which protect against frostbite. Moreover ice melts, therefore it is also important to be careful
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        <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">Miniprep</a></li>
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                not to place the iced object near electric devices. There is a risk of electrocution.</div></td></tr>
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     </ul>
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        </table>
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</section>
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    </center>
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<section>
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    <br/>
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     <h2>Gel migration</h2>
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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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     <ul>
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    <br/>
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         <li><a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">Gel electrophoresis</a></li>
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    <center>
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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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         <table class="tableau">
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    </ul>
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                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
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</section>
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                <tr><td><div class="petit">During this step, cells are manipulated. It is thus necessary
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<section>
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                to work in the sterile environment provided by the flow hood.</div></td></tr>
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     <h2>Test techniques</h2>
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        </table>
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     <ul>
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     </center>
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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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    <br/>
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     </ul>
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    <li>Let sit for 30 minutes on ice.</li>
 +
     <br/>
 +
    <li>Set the heating block at 42°C.</li>
 +
     <br/>
 +
    <center>
 +
         <table class="tableau">
 +
                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
 +
                <tr><td><div class="petit">It is important to know how a device works before using it.  
 +
The heating block takes a long time before reaching a defined temperature. Therefore,
 +
                it is recommended to switch it on a while before you need it. It is possible to accelerate the temperature increase by setting the device to
 +
a higher temperature than the one required. Of course it has to be set to 42∞C. In case the protocol requires a higher, the
 +
                users must be careful of the electric cables. They must not touch the hot spot of the device.
 +
                Users must also not forget to stop the device once it is not needed any further.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
 +
    <li>Incubate cells for 30 seconds at 42°C.</li>
 +
    <br/>
 +
    <center>
 +
         <table class="tableau">
 +
                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
 +
                <tr><td><div class="petit">Make sure that the eppendorfs are closed.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
 +
    <li>Incubate cells on ice for 2 min.</li>
 +
    <br/>
 +
    <li>Add 1 mL of LB medium at room temperature.</li>
 +
    <br/>
 +
    <center>
 +
        <table class="tableau">
 +
                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
 +
                <tr><td><div class="petit">This has to be done in a sterile area.</div></td></tr>
 +
        </table>
 +
    </center>
 +
     <br/>
 +
    <li>Incubate for 1 hour at 37°C on shaker.</li>
 +
    <br/>
 +
     <li>Spread 100-300 µL onto a plate complemented with appropriate antibiotic.</li>
 +
    <br/>
 +
     <center>
 +
         <table class="tableau">
 +
                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
 +
                <tr><td><div class="petit">This has to be done in a sterile environment.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
 +
     <li>Grow overnight at 37°C.</li>
 +
     <br/>
 +
    <center>
 +
         <table class="tableau">
 +
                <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
 +
                <tr><td><div class="petit">When leaving devices on working devices in the laboratory, users
 +
                must tell the person in charge of the laboratory. In addition, they must place a note on the device in
 +
                order to inform the personnal of the laboratory that an experiment is in progress.
 +
                </div></td></tr>
 +
        </table>
 +
     </center>
 +
</ol>
</section>
</section>
</div>
</div>

Latest revision as of 08:22, 25 September 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

Transformation of E. coli competent cells (heat shock)

Goal

Transform E. coli competent cells with foreign DNA.

Protocol

Following are the instructions from the OpenWetWare website.

  1. Prepare ice containers.

    2. SAFETY AND USEFUL RECOMMANDATION
    To prevent the ice container from falling off the workbench, it is recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack. It is important to know where manipulations will be achieved in order to apprehend the possible risks.

  2. Thaw TSS E. coli competent cells (from the freezer) on ice.

    3. SAFETY AND USEFUL RECOMMANDATION
    Using the -80°C freezer requires to wear special gloves which protect against frostbite. Moreover ice melts, therefore it is also important to be careful not to place the iced object near electric devices. There is a risk of electrocution.

  3. Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).

    4. SAFETY AND USEFUL RECOMMANDATION
    During this step, cells are manipulated. It is thus necessary to work in the sterile environment provided by the flow hood.

  4. Let sit for 30 minutes on ice.

  5. Set the heating block at 42°C.

    6. SAFETY AND USEFUL RECOMMANDATION
    It is important to know how a device works before using it. The heating block takes a long time before reaching a defined temperature. Therefore, it is recommended to switch it on a while before you need it. It is possible to accelerate the temperature increase by setting the device to a higher temperature than the one required. Of course it has to be set to 42∞C. In case the protocol requires a higher, the users must be careful of the electric cables. They must not touch the hot spot of the device. Users must also not forget to stop the device once it is not needed any further.

  6. Incubate cells for 30 seconds at 42°C.

    7. SAFETY AND USEFUL RECOMMANDATION
    Make sure that the eppendorfs are closed.

  7. Incubate cells on ice for 2 min.

  8. Add 1 mL of LB medium at room temperature.

    9. SAFETY AND USEFUL RECOMMANDATION
    This has to be done in a sterile area.

  9. Incubate for 1 hour at 37°C on shaker.

  10. Spread 100-300 µL onto a plate complemented with appropriate antibiotic.

    11. SAFETY AND USEFUL RECOMMANDATION
    This has to be done in a sterile environment.

  11. Grow overnight at 37°C.

    12. SAFETY AND USEFUL RECOMMANDATION
    When leaving devices on working devices in the laboratory, users must tell the person in charge of the laboratory. In addition, they must place a note on the device in order to inform the personnal of the laboratory that an experiment is in progress.

Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2"