Difference between revisions of "Team:NYU Shanghai/Protocols"
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} | } | ||
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+ | font-family: "helveticaNL"; | ||
+ | text-decoration: none; | ||
+ | color: #6db; | ||
+ | } | ||
+ | #PCR a { | ||
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<li>Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm. </li> | <li>Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm. </li> | ||
<li>Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.</li> | <li>Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.</li> | ||
+ | <li>Check results on Nanodrop.</li> | ||
</ol> | </ol> | ||
<br> | <br> | ||
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<br><br>Materials | <br><br>Materials | ||
<br>MinElute Midi Gel Extraction Kit | <br>MinElute Midi Gel Extraction Kit | ||
− | + | <br>Procedure | |
<ol> | <ol> | ||
<li>Pre-weigh a 2.0mL microcentrifuge tube</li> | <li>Pre-weigh a 2.0mL microcentrifuge tube</li> | ||
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<br> | <br> | ||
<div id="PCRText" style="display:none"> | <div id="PCRText" style="display:none"> | ||
− | + | <p>These are the conditions we used to PCR the gBlocks received from IDT. We used <a href="http://www.snapgene.com"> Snapgene </a> to design the primers. We used <a href="https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491">Q5 High-Fideltiy Polymerase</a> from New England Biolabs. | |
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <td>Component</td> | ||
+ | <td>50uL Reaction</td> | ||
+ | <td>Concentration</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5X Q5 Reaction Mix</td> | ||
+ | <td>25uL</td> | ||
+ | <td>1X</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10X NEB Buffer</td> | ||
+ | <td>5uL (1X)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10uM Forward primer</td> | ||
+ | <td>2.5uL</td> | ||
+ | <td>0.5uM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10uM Forward primer</td> | ||
+ | <td>2.5uL</td> | ||
+ | <td>0.5uM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Template DNA</td> | ||
+ | <td>10uL</td> | ||
+ | <td>100ng</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Nuclease-free water</td> | ||
+ | <td>10uL or none</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 02:40, 17 September 2015