Team:Macquarie Australia/WetLab/Protocols/PCR

From 2014.igem.org

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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
 +
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Operons">Induction of Operons</a></li>
</ul>
</ul>
</div>
</div>
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<li>1 µL of reverse primer.</li>
<li>1 µL of reverse primer.</li>
<li>1 µL of template.</li>
<li>1 µL of template.</li>
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<li>Total volume = 20 µL</li>
</ul>
</ul>
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<h4>Total volume = 20 µL</h4>
 
<h4>PCR settings: A general program for PCR is as follows:</h4>
<h4>PCR settings: A general program for PCR is as follows:</h4>
<ul>
<ul>
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<li>Initial denaturation at 98<sub>o</sub>C for 30seconds.</li>
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<li>Initial denaturation at 98<sup>o</sup>C for 30seconds.</li>
</ul>
</ul>
<h4>Followed by 30 repeats of:</h4>
<h4>Followed by 30 repeats of:</h4>
<ul>
<ul>
-
<li>Denaturation – 98oC for 10seconds.</li>
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<li>Denaturation – 98<sup>o</sup>C for 10seconds.</li>
-
<li>Annealing – 60<sub>o</sub>C for 10seconds.</li>
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<li>Annealing – 60<sup>o</sup>C for 10seconds.</li>
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<li>Extension – 72<sub>o</sub>C for 2minutes.</li>
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<li>Extension – 72<sup>o</sup>C for 2minutes.</li>
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<li>Final extension – 72<sub>o</sub>C for 10minutes.</li>
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<li>Final extension – 72<sup>o</sup>C for 10minutes.</li>
</ul>
</ul>

Latest revision as of 13:10, 17 October 2014

Protocols


PCR


PCR Mixture: General recipe for PCR is as follows:

  • 4 µL of 5x phusion buffer.
  • 0.4 µL dNTPs.
  • 0.6 µL of DMSO.
  • 0.2 µL of polymerase.
  • 11.8 µL of water.

To this mixture, add:

  • 1 µL of forward primer.
  • 1 µL of reverse primer.
  • 1 µL of template.
  • Total volume = 20 µL

PCR settings: A general program for PCR is as follows:

  • Initial denaturation at 98oC for 30seconds.

Followed by 30 repeats of:

  • Denaturation – 98oC for 10seconds.
  • Annealing – 60oC for 10seconds.
  • Extension – 72oC for 2minutes.
  • Final extension – 72oC for 10minutes.

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