Team:Grenoble/Biology/Protocols/Transformation 2

From 2012.igem.org

(Difference between revisions)
Line 7: Line 7:
     <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 with foreign DNA.
+
     Transform <i>E. coli</i> competent cells whith foreign DNA.
</section>
</section>
<br/>
<br/>
Line 17: Line 17:
<br/>
<br/>
<ol>
<ol>
-
     <li>Thaw TSS cells on ice.</li>
+
    <li>Prepare ice backs.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">In order to reduce the risk of ice that could fall, it
 +
                is recommended to place the back near the place manipulations and in case of any move,
 +
                it is better to take eppendorfes in an eppendorf rack. That is why it is important to
 +
                know where manipulations will be done.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
 +
     <li>Thaw TSS cells (from the freezer) on ice.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">When doing that it is necessary to wear special gloves
 +
                which protect against frostbite. Indeed cells are in a freezer where the temperature
 +
                is around -80°C. Moreover the ice melts, therefore it is also important to be careful
 +
                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>
 +
    </center>
 +
    <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>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">During this step, cells are manipulated. It is then necessary
 +
                to work in a sterile spot, by using a flow hood.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <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/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">It is important to know before manipulating how the device has to
 +
                be used. The heating block takes à long time before reaching the good temperature. Therefore
 +
                it is recommended to ask the device to reach a temperature higher than the one that has to be
 +
                reached for the manipulation. Asking a temperature of 53°C at the beginning accelerates 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 also not forget to stop the device once it is not used anymore.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
     <li>Incubate cells for 30 seconds at 42°C.</li>
     <li>Incubate cells for 30 seconds at 42°C.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">Users must pay attention that eppendorfs are well closed.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
     <li>Incubate cells on ice for 2 min.</li>
     <li>Incubate cells on ice for 2 min.</li>
     <li>Add 1 mL of LB medium at room temperature.</li>
     <li>Add 1 mL of LB medium at room temperature.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">This has to be done in a sterile spot.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <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>
     <li>Spread 100-300 µL onto a plate made with appropriate antibiotic.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">This has to be done in a sterile spot.</div></td></tr>
 +
        </table>
 +
    </center>
 +
    <br/>
     <li>Grow overnight at 37°C.</li>
     <li>Grow overnight at 37°C.</li>
 +
    <br/>
 +
    <center>
 +
        <table>
 +
                <tr><th><i><div class="petit">SAFETY AND USEFUL RECOMMANDATION</div></i></th></tr>
 +
                <tr><td><div class="petit">When leaving things in the lab that keep on working, users
 +
                must tell the person in charge of the lab and placing an inscription on the device in
 +
                order to not have someone who turns the device off during the absence of the other manipulators.
 +
                </div></td></tr>
 +
        </table>
 +
    </center>
</ol>
</ol>
</section>
</section>

Revision as of 19:01, 27 August 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 whith foreign DNA.

Protocol

Following are the instructions from the OpenWetWare website.

  1. Prepare ice backs.

    2. SAFETY AND USEFUL RECOMMANDATION
    In order to reduce the risk of ice that could fall, it is recommended to place the back near the place manipulations and in case of any move, it is better to take eppendorfes in an eppendorf rack. That is why it is important to know where manipulations will be done.

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

    3. SAFETY AND USEFUL RECOMMANDATION
    When doing that it is necessary to wear special gloves which protect against frostbite. Indeed cells are in a freezer where the temperature is around -80°C. Moreover the ice melts, therefore it is also important to be careful to not place ice near electric devices. There is a risk of electrocution. To use cells the substance must have melt.

  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 then necessary to work in a sterile spot, by using a flow hood.

  4. Let sit for 30 minutes on ice.
  5. Prepare the heating block for a temperature of 42°C.

    6. SAFETY AND USEFUL RECOMMANDATION
    It is important to know before manipulating how the device has to be used. The heating block takes à long time before reaching the good temperature. Therefore it is recommended to ask the device to reach a temperature higher than the one that has to be reached for the manipulation. Asking a temperature of 53°C at the beginning accelerates 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 also not forget to stop the device once it is not used anymore.

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

    7. SAFETY AND USEFUL RECOMMANDATION
    Users must pay attention that eppendorfs are well 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 spot.

  9. Incubate for 1 hour at 37°C on shaker.
  10. Spread 100-300 µL onto a plate made with appropriate antibiotic.

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

  11. Grow overnight at 37°C.

    12. SAFETY AND USEFUL RECOMMANDATION
    When leaving things in the lab that keep on working, users must tell the person in charge of the lab and placing an inscription on the device in order to not have someone who turns the device off during the absence of the other manipulators.

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