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iGEM Grenoble 2012


Gibson Assembly


Ligate DNA fragments.

Mix preparation

Following are the instructions from the Synbio website.


Gibson Assembly reaction

Following are the instructions from the Synbio website.

  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.

    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 to dry it after. Be careful to use for each sample a different sterile cone. It is also important to well close tubes after putting the ingredient in. Moreover the DNA cannot resist until it is not incorporated in a plasmid and into a cell. Therefore there is no chance that there are any possible consequences because of a leakage in the environment.

  3. Put the 50 µL PCR tube into the thermocycler.

  4. Turn on the device.

    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 cause risks for thermocycler users. Indeed a thermocycler is an electric device which can become 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 instruction before using it. In our group we managed to learn how to use it before doing any manipulation. Besides on the device there are also protections to prevent accident.