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Team:TU Delft/protocols/PCR
From 2010.igem.org
PCR
Materials:
- Pfx Polymerase (Invitrogen)
- 10x Pfx Bufer (Invitrogen)
- Enhancer (Invitrogen)
- 50 mM MgSO4
- 10 mM dNTPs
- Primer solutions 5 pmol/μL
- Template DNA (plasmid at 50 pg – 1 ng/μL)
Protocol:
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice. Preparation of reaction mixture:
1. Gently vortex and briefly centrifuge all solutions after thawing
2. Keep solutions on ice
3. Add to a thin walled PCR tube, on ice, for a 100 μL reaction:
1× pre-mix
| Component | Sample |
| Pfx polymerse | 0.6 μL |
| 10x Pfx buffer | 5 μL |
| Enhancer | 5 μL |
| 10 mM dNTPs | 1.5 μL |
| 50 mM MgSO4 | 1 μL |
| Primer 1 | 3 μL |
| Primer 2 | 3 μL |
| DNA template | 1 μL |
| H20 | 29.9 μL |
4. Gently vortex the sample and briefly centrifuge (5 sec) to collect all droplets at the bottom of the tube
5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.
PCR program:
| Step | Annealing Temperature | Time, min:sec | Number of cycles |
| Initial denaturation | x °C * | 2:00 | 1 |
| Annealing | 95 °C | 2:00 | 1 |
| Extension | 68 °C | 1:00 | 1 |
| Denaturation | 95 °C | 1:00 | 25 |
| Annealing | x °C * | 1:00 | 25 |
| Extension | 68 °C | 1:00 | 25 |
| Final Extension | 68 °C | 10:00 | 1 |
* Calculate the optimal annealing temperature = 3x G/C + 2x A/T
