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Team:Macquarie Australia/WetLab/Protocols/PCR
From 2014.igem.org
(Difference between revisions)
| Line 47: | Line 47: | ||
<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> | ||
| - | <li>Initial denaturation at 98< | + | <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 – | + | <li>Denaturation – 98<sup>o</sup>C for 10seconds.</li> |
| - | <li>Annealing – 60< | + | <li>Annealing – 60<sup>o</sup>C for 10seconds.</li> |
| - | <li>Extension – 72< | + | <li>Extension – 72<sup>o</sup>C for 2minutes.</li> |
| - | <li>Final extension – 72< | + | <li>Final extension – 72<sup>o</sup>C for 10minutes.</li> |
</ul> | </ul> | ||
Revision as of 05:20, 16 October 2014
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.
