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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     Assembling two DNA fragments.
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     Ligate two DNA fragments <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">cut using restriction enzymes</a>.
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</section>
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     <h2>Protocol</h2>
     <h2>Protocol</h2>
       Following are the instructions from the
       Following are the instructions from the
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<a href="http://www.neb.com/nebecomm/products/protocol658.asp">NEB website</a>.
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<a href="http://www.neb.com/nebecomm/products/protocol658.asp" target="_blank">NEB website</a>.
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<br/>
<br/>
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<ol>
<ol>
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    <li>Set up the water bath at 16°C.</li>
 
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    <br/>
 
     <li>In an eppendorf tube, introduce : <ul><div class="petit">
     <li>In an eppendorf tube, introduce : <ul><div class="petit">
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         <li>1 µL of T4 DNA ligase</li>
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         <li>1 µL of <a href="http://www.neb.com/nebecomm/products/productM0202.asp" target="_blank">T4 DNA ligase</a></li>
         <li>2 µL of T4 DNA ligase buffer (10X)</li>
         <li>2 µL of T4 DNA ligase buffer (10X)</li>
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         <li>4 µL of DNA vector</li>
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         <li>4 µL (50 ng) of DNA vector</li>
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         <li>12 µL of DNA insert</li>
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         <li>12 µL (50 ng) of DNA insert</li>
         <li>to 20 µL of water</li>
         <li>to 20 µL of water</li>
         </div>
         </div>
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     <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
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                 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
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                 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
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                 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
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                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>
     <br/>
     <br/>
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    <li>Put the eppendorf tube into the water bath.</li>
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     <li>Incubate at room temperature for 10 minutes.</li>
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    <br/>
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     <li>Incubate at 16°C for 10 minutes.</li>
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</ol>
</ol>
</section>
</section>

Latest revision as of 08:20, 25 September 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
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.

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