Difference between revisions of "Team:NAIT Edmonton/Protocols"

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        <center><h1>Ligation of Vectors and Inserts</h1></center>
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      <br>
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1. Set up the following reaction mixture on ice:
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<table class="tg">
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<tr>
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<strong>
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<th class="tg-hgcj">Component</th>
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<th class="tg-hgcj">20µL Reaction</th>
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</strong>
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</tr>
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<tr>
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<td class="tg-s6z2">10X T4 Ligase Buffer</td>
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<td class="tg-s6z2">2µL</td>
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</tr>
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<tr>
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<td class="tg-s6z2">Vector DNA (4kb)</td>
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<td class="tg-s6z2">50ng (0.020pmol)</td>
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</tr>
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<tr>
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<td class="tg-s6z2">Insert DNA (1kb)</td>
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<td class="tg-s6z2">37.5ng (0.60pmol)</td>
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</tr>
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<tr>
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<td class="tg-s6z2">Nuclease Free H<sub>2</sub>O</td>
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<td class="tg-s6z2">Up to 20µL</td>
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</tr>
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<tr>
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<td class="tg-s6z2">T4 DNA Ligase</td>
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<td class="tg-s6z2">1µL</td>
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</tr>
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</table>
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2. Gently mix the reaction by pipetting up and down and microfuge briefly<br>
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3. For cohesive ends, incubate at 16°C overnight or at room temperature for 10 minutes<br>
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4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours<br>
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5. Heat inactivate at 65°C for 10 minutes<br>
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6. Chill on ice and transform 1-5 µL if the reaction into 50 µL competent Cells<br>
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  </div>
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Revision as of 01:57, 16 September 2015

Team NAIT 2015

Experimental Design

Go through our interactive experimental design flow chart! Many of our protocols are manufacturer specicfied; however, some are customized by us! Click on the flow chart boxes if the hand cursor appears to read more about our customized protocols.

PDFs of protocols can also be found

Theorizing our Sequences
Literature has shown certain proteins inherently stain in colour.
Looked up characteristics of said proteins
Isolated and identified the unique characteristics of said proteins so that we can manually write our own sequences and generate custom proteins.
Writing our Sequences
Went down to base pair level and wrote out our sequences with the defining characteristics identified in literature.
PCR
Digestion and Ligation
Transforming Bacteria
Validating the Transformation
Protein Isolation and Purification
SDS PAGE and Silver Staining