Difference between revisions of "Team:NYU Shanghai/Protocols"
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<br> | <br> | ||
<div id="ligateText" style="display:none"> | <div id="ligateText" style="display:none"> | ||
− | + | <p>Link to <a href="https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202"> NEB Protocol </a></p> | |
+ | <ol> | ||
+ | <li>Make Reaction mixture</li> | ||
+ | <table border="1"> | ||
+ | <tr style="font-weight: bold;"> | ||
+ | <td>COMPONENTS</td> | ||
+ | <td>20 μl REACTION</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10X T4 DNA Ligase Buffer*</td> | ||
+ | <td>2 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Vector DNA (4 kb)</td> | ||
+ | <td>50 ng (0.020 pmol)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Insert DNA (1 kb)</td> | ||
+ | <td>37.5 ng (0.060 pmol)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Nuclease-free water</td> | ||
+ | <td>to 20 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1 μl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <li>Controls</li> | ||
+ | <ul> | ||
+ | <li>Reaction mixture with no insert DNA</li> | ||
+ | <li>Reaction mixture with no insert DNA and no ligase</li> | ||
+ | </ul> | ||
+ | <li>Ligation temperature and times vary</li> | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <td>For inserting a part into backbone (no 3A assembly), the suggested NEB protocol worked</td> | ||
+ | <td>16C overnight or room temperature 10min</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>For 3A Assembly</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <li>Heat inactivate at 65°C for 10 minutes.</li> | ||
+ | <li>Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.</li> | ||
+ | </ol> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 11:51, 17 September 2015