Team:Grenoble/Biology/Protocols/GA

From 2012.igem.org

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     <h1>Gibson Assembly</h1>
     <h1>Gibson Assembly</h1>
     <h2>Goal</h2>
     <h2>Goal</h2>
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     Ligate DNA fragments.
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     Assemble DNA fragments.
</section>
</section>
<br/>
<br/>
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     <h2>Mix preparation</h2>
     <h2>Mix preparation</h2>
     Following are the instructions from the
     Following are the instructions from the
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<a href="http://synbio.org.uk/dna-assembly/guidetogibsonassembly.html">Synbio website</a>.
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<a href="http://synbio.org.uk/dna-assembly/guidetogibsonassembly.html" target="blank">Synbio website</a>.
<br/>
<br/>
<br/>
<br/>
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     <br/>
     <br/>
     <center>
     <center>
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         <table>
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         <table class="tableau">
                 <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
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                 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
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                 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
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                 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
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                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>
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                consequences because of a leakage in the environment.</div></td></tr>
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         </table>
         </table>
     </center>
     </center>
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     <br/>
     <br/>
     <center>
     <center>
-
         <table>
+
         <table class="tableau">
                 <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">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,
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                 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
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                learn how to use it before doing any manipulation. Besides on the device there are also protections to prevent
+
                 accidents.</div></td></tr>
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                 accident.</div></td></tr>
+
         </table>
         </table>
     </center>
     </center>
 +
</section>
 +
<br/>
 +
<section>
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<h2>References</h2>
 +
<ul>
 +
<li>Gibson D. G. (2009). One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. <i>Nature Protocols</i>.</li>
 +
<br/>
 +
<li>Gibson D. G., Young L., Chuang R-Y, Venter J. C., Hutchison C. A., Smith H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature Methods</i>. Vol. 6 No. 5.</li>
</section>
</section>
</div>
</div>

Latest revision as of 08:21, 25 September 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

Gibson Assembly

Goal

Assemble DNA fragments.

Mix preparation

Following are the instructions from the Synbio website.

GA_mix

Gibson Assembly reaction

  1. Set up the thermocycler at 50°C for one hour.

  2. In a 50 µL PCR tube, introduce :
    • 15 µL of Master Mix (1.33 X)
    • 5 µL of DNA fragments
    Optimized cloning efficiency is 50-100 ng of vectors with 2-3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps.

    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 50 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.

References

  • Gibson D. G. (2009). One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. Nature Protocols.

  • Gibson D. G., Young L., Chuang R-Y, Venter J. C., Hutchison C. A., Smith H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. Vol. 6 No. 5.

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