Team:Grenoble/Biology/Protocols/PCR 2

From 2012.igem.org

(Difference between revisions)
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     <h1>PCR using a HF Phusion polymerase</h1>
     <h1>PCR using a HF Phusion polymerase</h1>
     <h2>Goal</h2>
     <h2>Goal</h2>
-
     Amplify a section of DNA.
+
     Amplify a DNA fragment.
</section>
</section>
<br/>
<br/>
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<br/>
<br/>
<ol>
<ol>
-
     <li>Set up the thermocycler at the right temperatures.</li>
+
     <li>Set the thermocycler at the right temperatures.</li>
     <br/>
     <br/>
-
     <li>In a 50 µL PCR tube, introduce : <ul><div class="petit">
+
     <li>In a 200 µL PCR tube, introduce : <ul><div class="petit">
-
         <li>1.25 µL of forward primer (10µM)</li>
+
         <li>1.25 µL of forward primer</li>
-
         <li>1.25 µL of reverse primer (10µM)</li>
+
         <li>1.25 µL of reverse primer</li>
         <li>2.5 µL of dNTP (2 mM)</li>
         <li>2.5 µL of dNTP (2 mM)</li>
         <li>5 µL of buffer</li>
         <li>5 µL of buffer</li>
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                 <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></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">Those products are not dangerous at first sight, but in case of spreading
                 <tr><td><div class="petit">Those products are not dangerous at first sight, but in case of spreading
-
                 or in case of products on hands or on any parts on the body, it is recommended to wash this part and
+
                 or contacts with any part of the body, it is recommended to wash this part and
-
                 to dry it after.  Be careful to use for each sample a different sterile cone. It is also important to
+
                 to dry it after.  Use a different pipette tip for each sample. It is also important to
-
                 well close tubes after putting the ingredient in. Moreover the DNA cannot resist until it is not
+
                 close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced
-
                incorporated in a plasmid and into a cell. Therefore there is no chance that there are any possible
+
                into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.</div></td></tr>
-
                consequences because of a leakage in the environment.</div></td></tr>
+
         </table>
         </table>
     </center>
     </center>
     <br/>
     <br/>
-
     <li>Put the 50 µL PCR tube into the thermocycler.</li>
+
     <li>Put the 200 µL PCR tube into the thermocycler.</li>
     <br/>
     <br/>
     <li>Turn on the device.</li>
     <li>Turn on the device.</li>
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                 <tr><td><div class="petit">It was noticed that it exists a certain number of different thermocycler models.
                 <tr><td><div class="petit">It was noticed that it exists a certain number of different thermocycler models.
                 That is why biologists do not know, all the time, how the device they will use, has to be manipulated. This
                 That is why biologists do not know, all the time, how the device they will use, has to be manipulated. This
-
                 can cause risks for thermocycler users. Indeed a thermocycler is an electric device which can become hot,
+
                 can present risks for thermocycler users. Indeed a thermocycler is an electric device which can become very hot,
-
                 and in case of misused people can be injured. Consequently it is very important for users to know how to use
+
                 and in case of misused, people can be injured. Consequently it is very important for users to know how to use
-
                 the device. It is recommended to read the operating instruction before using it. In our group we managed to
+
                 the device. It is recommended to read the operating instructions before using it. Besides, the device has protective measures to prevent
-
                learn how to use it before doing any manipulation. Besides on the device there are also protections to prevent
+
                 accidents.</div></td></tr>
-
                 accident.</div></td></tr>
+
         </table>
         </table>
     </center>
     </center>

Revision as of 14:41, 3 September 2012

iGEM Grenoble 2012

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Project

PCR using a HF Phusion polymerase

Goal

Amplify a DNA fragment.

Protocol

Following are the instructions from the NEB website.

  1. Set the thermocycler at the right temperatures.

  2. In a 200 µL PCR tube, introduce :
    • 1.25 µL of forward primer
    • 1.25 µL of reverse primer
    • 2.5 µL of dNTP (2 mM)
    • 5 µL of buffer
    • 0.75 µL of DMSO (optional)
    • variable volume of template DNA
    • 0.25 µL of HF Phusion polymerase
    • to 25 µL of nuclease-free water

    SAFETY AND USEFUL RECOMMANDATION
    Those products are not dangerous at first sight, but in case of spreading or contacts with any part of the body, it is recommended to wash this part and to dry it after. Use a different pipette tip for each sample. It is also important to close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.

  3. Put the 200 µL PCR tube into the thermocycler.

  4. Turn on the device.

    5. SAFETY AND USEFUL RECOMMANDATION
    It was noticed that it exists a certain number of different thermocycler models. That is why biologists do not know, all the time, how the device they will use, has to be manipulated. This can present risks for thermocycler users. Indeed a thermocycler is an electric device which can become very hot, and in case of misused, people can be injured. Consequently it is very important for users to know how to use the device. It is recommended to read the operating instructions before using it. Besides, the device has protective measures to prevent accidents.

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