Team:Grenoble/Biology/Protocols/Ligation

From 2012.igem.org

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     <h1>Ligation</h1>
     <h1>Ligation</h1>
     <h2>Goal</h2>
     <h2>Goal</h2>
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     Ligate two DNA fragments first <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">cut by restriction enzymes</a>.
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     Ligate two DNA fragments <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">cut using restriction enzymes</a>.
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</section>
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Revision as of 14:27, 20 September 2012

iGEM Grenoble 2012

Project

Ligation

Goal

Ligate two DNA fragments cut using restriction enzymes.

Protocol

Following are the instructions from the NEB website.

  1. In an eppendorf tube, introduce :
    • 1 µL of T4 DNA ligase
    • 2 µL of T4 DNA ligase buffer (10X)
    • 4 µL (50 ng) of DNA vector
    • 12 µL (50 ng) 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 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.

  2. Incubate at room temperature for 10 minutes.