Fong:PCR
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Preparation
- 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 smallest Tm of the primers.
- Use final extension time of 1 min.
- May use extended primary melting time, to ensure full denaturation of template.
- Use at least 30s to one min extension cycle per kb of product.
PCR Taq Mix
Name | Volume | |||
---|---|---|---|---|
Taq buffer | 2.5 μL | |||
dNTP | 0.5 μL | |||
F-primer (25nM) | 1.25 μL | |||
R-primer (25nM) | 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 |
Total V | 25 μL |
- 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!
- ????
- PROFT
- Be sure to run at least four varying Mg2+ concentrations in order to optimize yield and fidelity.
- Run a gel using some product and product digestion to verify fragment size and proper restriction sites.
PCR Phusion High-Fidelity Polymerase Mix
Name | Volume | ||
---|---|---|---|
Phusion buffer | 5 μL | ||
dNTP | 0.2 μL | ||
F-primer (25nM) | 0.5 μL | ||
R-primer (25nM) | 0.5 μL | ||
Phusion Polymerase | 0.25 μL | ||
DNA | 1 μL | ||
DMSO | 0.75 μL | ||
Mg2+ | 0 | 0.5 | 1.0 μL |
H2O | 16.8 | 16.3 | 15.8 μL |
Total V | 25 μL |
- Add all reagents to labeled PCR reaction tube, adding Phusion polymerase last.
- Place in the thermocycler and select cycle, making any changes as noted above.
- Run PCR reaction.
- Enjoy your brand-new DNA!
- ????
- PROFT
- Be sure to run varying Mg2+ concentrations in order to optimize yield and fidelity.
- Run a gel using some product and template to verify fragment size and proper restriction sites.