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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> | ||
| - | + | Ligate two DNA fragments <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">cut using restriction enzymes</a>. | |
</section> | </section> | ||
<br/> | <br/> | ||
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<h2>Protocol</h2> | <h2>Protocol</h2> | ||
Following are the instructions from the | Following are the instructions from the | ||
| - | <a href="http://www.neb.com/nebecomm/products/protocol658.asp">NEB website</a>. | + | <a href="http://www.neb.com/nebecomm/products/protocol658.asp" target="_blank">NEB website</a>. |
<br/> | <br/> | ||
<br/> | <br/> | ||
<ol> | <ol> | ||
| - | |||
| - | |||
<li>In an eppendorf tube, introduce : <ul><div class="petit"> | <li>In an eppendorf tube, introduce : <ul><div class="petit"> | ||
| - | <li>1 µL of T4 DNA ligase</li> | + | <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> | ||
| - | <li>4 µL of DNA vector</li> | + | <li>4 µL (50 ng) of DNA vector</li> |
| - | <li>12 µL of DNA insert</li> | + | <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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<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">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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</center> | </center> | ||
<br/> | <br/> | ||
| - | + | <li>Incubate at room temperature for 10 minutes.</li> | |
| - | + | ||
| - | <li>Incubate at | + | |
</ol> | </ol> | ||
</section> | </section> | ||
Latest revision as of 08:20, 25 September 2012
Ligation
Goal
Ligate two DNA fragments cut using restriction enzymes.Protocol
Following are the instructions from the NEB website.- 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.
- Incubate at room temperature for 10 minutes.
