Team:UC Davis/Protocols

From 2013.igem.org

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12 µL total
12 µL total
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<p class="menu_head">Measuring DNA Concentration</p>
<p class="menu_head">Measuring DNA Concentration</p>
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<li>Same as above.</li>
<li>Same as above.</li>
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</div>
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<p class="menu_head">Colony PCR</p>
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                <p>When to Use</p>
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<li>Transformation of competent cells may yield false positives. Through colony PCR, one may screen for the presence of the assembled sequence by amplifying with the universal forward and reverse primers and checking the length of the fragments through gel electrophoresis.</li>
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<p>Materials</p>
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<li>2 µL    10x Buffer</li>
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<li>4 µL    Q solution</li>
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<li>1 µL    Universal Forward Primer (0.1 µg/µL)</li>
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<li>1 µL    Universal Reverse Primer (0.1 µg/µL)</li>
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<li>0.5 µL  dNTPs (10 mM)</li>
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<li>1.375 µL    dH<sub>2</sub>O</li>
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<li>Pick a colony with a sterile tip and set the tip in 10 µL dH<sub>2</sub>O in a PCR tube for 10 minutes. Pipet up and down to resuspend the cell material and then add the reagent, DNA, and buffer mixture. While picking the colony, some cells should also be transferred to a new plate with the same antibiotic so that if the screen results are positive, the colony may be identified and grown up.</li>
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20 µL Total
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<br></br>
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<p>PCR program</p>
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<ol>
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<li>98º C      30 sec</li>
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<li>98º C      10 sec</li>
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<li>55º C      30 sec    </li>               
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<li>72º C      1 min / kb    Repeat Steps 2-4 29x (30x total)  </li>
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<li>72º C      5 min</li>
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<li>4º C      Hold </li>
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</ol>
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</div>

Revision as of 07:52, 24 September 2013

Protocols

Retrieved from "http://2013.igem.org/Team:UC_Davis/Protocols"