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>
     <h1>Transformation of <i>E. coli</i> competent cells (heat shock)</h1>
     <h2>Goal</h2>
     <h2>Goal</h2>
-
     Transform <i>E. coli</i> competent cells whith foreign DNA.
+
     Transform <i>E. coli</i> competent cells with foreign DNA.
</section>
</section>
<br/>
<br/>
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     <h2>Protocol</h2>
     <h2>Protocol</h2>
Following are the instructions from the
Following are the instructions from the
-
<a href="http://openwetware.org/wiki/Transforming_chemically_competent_cells">OpenWetWare website</a>.
+
<a href="http://openwetware.org/wiki/Transforming_chemically_competent_cells" target="blank">OpenWetWare website</a>.
<br/>
<br/>
<br/>
<br/>
<ol>
<ol>
-
     <li>Prepare ice backs.</li>
+
     <li>Prepare ice containers.</li>
     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
-
                 <tr><td><div class="petit">In order to reduce the risk of ice that could fall, it
+
                 <tr><td><div class="petit">To prevent the ice container from falling off the workbench, it is
-
                 is recommended to place the back near the place manipulations and in case of any move,
+
                 recommended to place it away from edges. In addition, it is better to put eppendorfs in a special rack.  
-
                it is better to take eppendorfes in an eppendorf rack. That is why it is important to
+
It is important to know where manipulations will be achieved in order to apprehend the possible risks.</div></td></tr>
-
                know where manipulations will be done.</div></td></tr>
+
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
-
     <li>Thaw TSS cells (from the freezer) on ice.</li>
+
     <li>Thaw <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">TSS <i>E. coli</i> competent cells</a> (from the freezer) on ice.</li>
     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
-
                 <tr><td><div class="petit">When doing that it is necessary to wear special gloves
+
                 <tr><td><div class="petit">Using the -80°C freezer requires to wear special gloves
-
                 which protect against frostbite. Indeed cells are in a freezer where the temperature
+
                 which protect against frostbite. Moreover ice melts, therefore it is also important to be careful
-
                is around -80°C. Moreover the 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.</div></td></tr>
-
                 to not place ice near electric devices. There is a risk of electrocution. To use cells
+
-
                the substance must have melt.</div></td></tr>
+
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
     <li>Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).</li>
     <li>Add DNA, pipette gently to mix (1 µL of prepped plasmid is more than enough).</li>
 +
    <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
-
                 <tr><td><div class="petit">During this step, cells are manipulated. It is then necessary
+
                 <tr><td><div class="petit">During this step, cells are manipulated. It is thus necessary
-
                 to work in a sterile spot, by using a flow hood.</div></td></tr>
+
                 to work in the sterile environment provided by the flow hood.</div></td></tr>
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
     <li>Let sit for 30 minutes on ice.</li>
     <li>Let sit for 30 minutes on ice.</li>
-
     <li>Prepare the heating block for a temperature of 42°C.</li>
+
    <br/>
 +
     <li>Set the heating block at 42°C.</li>
     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <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 before manipulating how the device has to
+
                 <tr><td><div class="petit">It is important to know how a device works before using it.
-
                be used. The heating block takes à long time before reaching the good temperature. Therefore
+
The heating block takes a long time before reaching a defined temperature. Therefore,
-
                 it is recommended to ask the device to reach a temperature higher than the one that has to be
+
                 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
-
                reached for the manipulation. Asking a temperature of 53°C at the beginning accelerates the
+
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
-
                increase of the temperature. But after that the temperature must be reduce to 42°C. The
+
-
                temperature here is not very high. But in case of manipulation asking for higher temperature,
+
                 users must be careful of the electric cables. They must not touch the hot spot of the device.
                 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 used anymore.</div></td></tr>
+
                 Users must also not forget to stop the device once it is not needed any further.</div></td></tr>
         </table>
         </table>
     </center>
     </center>
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     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
-
                 <tr><td><div class="petit">Users must pay attention that eppendorfs are well closed.</div></td></tr>
+
                 <tr><td><div class="petit">Make sure that the eppendorfs are closed.</div></td></tr>
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
     <li>Incubate cells on ice for 2 min.</li>
     <li>Incubate cells on ice for 2 min.</li>
 +
    <br/>
     <li>Add 1 mL of LB medium at room temperature.</li>
     <li>Add 1 mL of LB medium at room temperature.</li>
     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <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 spot.</div></td></tr>
+
                 <tr><td><div class="petit">This has to be done in a sterile area.</div></td></tr>
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
     <li>Incubate for 1 hour at 37°C on shaker.</li>
     <li>Incubate for 1 hour at 37°C on shaker.</li>
-
     <li>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li>
+
    <br/>
 +
     <li>Spread 100-300 µL onto a plate complemented with appropriate antibiotic.</li>
     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <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 spot.</div></td></tr>
+
                 <tr><td><div class="petit">This has to be done in a sterile environment.</div></td></tr>
         </table>
         </table>
     </center>
     </center>
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     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
-
                 <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
+
                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr>
-
                 <tr><td><div class="petit">When leaving things in the lab that keep on working, users
+
                 <tr><td><div class="petit">When leaving devices on working devices in the laboratory, users
-
                 must tell the person in charge of the lab and placing an inscription on the device in
+
                 must tell the person in charge of the laboratory. In addition, they must place a note on the device in
-
                 order to not have someone who turns the device off during the absence of the other manipulators.
+
                 order to inform the personnal of the laboratory that an experiment is in progress.
                 </div></td></tr>
                 </div></td></tr>
         </table>
         </table>

Latest revision as of 08:22, 25 September 2012

iGEM Grenoble 2012

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.

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

  4. 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.

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

  6. 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.

  7. Let sit for 30 minutes on ice.

  8. Set the heating block at 42°C.

  9. 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.

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

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

  12. Incubate cells on ice for 2 min.

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

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

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

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

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

  18. Grow overnight at 37°C.

  19. 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.