Team:UGA-Georgia/Protocols
From 2014.igem.org
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<li>Annealing temperature = 65 Celsius </li> | <li>Annealing temperature = 65 Celsius </li> | ||
<li>Run products on a gel for verification (2.5 uL of each)</li> | <li>Run products on a gel for verification (2.5 uL of each)</li> | ||
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+ | </ol> | ||
+ | </p> | ||
+ | </td> | ||
+ | |||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Plasmid Extraction</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="45%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>Add 1000 uL of culture to each centrifuge tube</li> | ||
+ | <li>Centrifuge tubes at 15000 rpm for 30 seconds</li> | ||
+ | <li>Dispose supernatant in biohazard bin</li> | ||
+ | <li>Add another 1000 uL of each culture to respective centrifuge tubes</li> | ||
+ | <li>Centrifuge again at 15000 rpm for 30 seconds</li> | ||
+ | <li>Discard supernatant in biohazard bin</li> | ||
+ | <li>Add 600 uL of DI water to each centrifuge tube</li> | ||
+ | <li>Vortex tubes to resuspend the pellet into the water</li> | ||
+ | <li>Add 100 uL of 7x lysis buffer to each tube and mix for 30 seconds</li> | ||
+ | <li>Add 350 uL of neutralization buffer and mix</li> | ||
+ | <li>Centrifuge at 15000 rpm for 3 minutes</li> | ||
+ | <li>Transfer supernatant to spin column</li> | ||
+ | <li>Centrifuge at 15000 rpm for 30 seconds</li> | ||
+ | <li>Discard liquid in collection tube</li> | ||
+ | <li>Add 200 uL of endo wash buffer to each spin column</li> | ||
+ | <li>Centrifuge at 15000 rpm for 30 seconds</li> | ||
+ | <li>Add 400 uL of zippy wash buffer to each spin column</li> | ||
+ | <li>Centrifuge at 15000 rpm for 1 minute</li> | ||
+ | <li>Place spin columns in centrifuge tubes</li> | ||
+ | <li>Add 40 uL of DI water to each column</li> | ||
+ | <li>Let stand for 2 minutes (to allow the gene and plasmid to dissolve in the water)</li> | ||
+ | <li>Centrifuge at 15000 rpm for 30 seconds</li> | ||
+ | <li>Store at -20 Celsius</li> | ||
+ | <li>Dispose of spin columns</li> | ||
+ | |||
</ol> | </ol> |
Revision as of 00:36, 13 October 2014
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Plasmid Extraction | ||||
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