Fong:PCR: Difference between revisions
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Revision as of 11:03, 25 June 2009
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Deng's PCR Protocol
Preparation of DNA
- Prepare DNA from cell sample during mid-log phase, or as extraction kit designates.
- Use Spectrophotometer to determine concentration and total amount of extracted DNA.
Program Thermocycler
- Use standard program.
- Use Tannealing that is 5°C lower than the least Tm of the primers.
- Use final extension time of 1:00.
- May use extended primary melting time, to ensure full denaturation of template.
PCR Reaction Mix
Name | Volume | |||
---|---|---|---|---|
Taq buffer | 2.5 μL | |||
dNTP | 0.5 μL | |||
F-primer | 1.25 μL | |||
R-primer | 1.25 μL | |||
Mg2+ | 0 | 0.5 | 1.0 | 1.5 |
Taq | 0.2 | 0.2 | 0.2 | 0.2 |
H2O | 17.3 | 16.8 | 16.3 | 15.8 |
DNA | 2 | 2 | 2 | 2 |
- Add all reagents to labeled PCR reaction tube, adding Taq polymerase last.
- Place in the thermocycler and select cycle, making any changes as noted above.
- Run PCR reaction.
- Enjoy your brand-new DNA!
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- PROFT