Team:Grenoble/Biology/Protocols/Ligation

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iGEM Grenoble 2012

Project

Ligation

Goal

Assembling two DNA fragments.

Protocol

Following are the instructions from the NEB website.

  1. Set up the water bath at 16°C.

  2. In an eppendorf tube, introduce :
    • 1 µL of T4 DNA ligase
    • 2 µL of T4 DNA ligase buffer (10X)
    • 4 µL of DNA vector
    • 12 µL of DNA insert
    • to 20 µL of water

    SAFETY AND USEFUL RECOMMANDATION
    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 eppendorf tube into the water bath.

  4. Incubate at 16°C for 10 minutes.