Team:SCU-China/Team

From 2014.igem.org

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<p>For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows:</p><table class="table table-striped"> <caption>PCR system (For 50&#956;L)</caption> <thead><tr><th><p>Materials</p>
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</th><th><p>Volume</p>
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</tr></thead> <tbody><tr><td><p>Taq DNA polymerase</p>
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</td><td><p>0.5&#956;L</p>
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</tr><tr><td><p>Template</p>
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</td><td><p>0.5&#956;L</p>
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</tr><tr><td><p>Forward Primer</p>
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</td><td><p>1&#956;L</p>
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</tr><tr><td><p>Reverse Primer</p>
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</td><td><p>1&#956;L</p>
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</tr><tr><td><p>dNTPs (2.5mM)</p>
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</td><td><p>4&#956;L</p>
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</tr><tr><td><p>10x Buffer (Mg2+ free)</p>
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</td><td><p>5&#956;L</p>
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</tr><tr><td><p>MgCl2 (25mM)</p>
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</td><td><p>3&#956;L</p>
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</tr><tr><td><p>Sterilized diluted water</p>
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</td><td><p>35&#956;L</p>
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</table><p>Note: The content of template should be less than 500ng for the 50&#956;L system. For all of our experiments, the content of template of 0.5&#956;L DNA lower than 500ng.</p>
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<table class="table table-striped"> <caption>PCR protocol</caption><thead><tr><th><p>Temperature</p>
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</th><th><p>Times</p>
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</tr></thead> <tbody><tr><td><p>95&#8451;</p>
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</td><td><p>5min.</p>
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</td><th rowspan="3">30-35 Times</th>
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</td><td><p>30sec.</p>
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</tr><tr><td><p>55-65&#8451;</p>
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</tr><tr><td><p>72&#8451;</p>
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</td><td><p>1-2min.</p>
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</tr><tr><td><p>72&#8451;</p>
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</td><td><p>10min.</p>
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</table><p>Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute.</p>
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Revision as of 05:26, 17 October 2014

Team

Sichuan university

Retrieved from "http://2014.igem.org/Team:SCU-China/Team"