| Protocol: PCR Reaction Setup | |
| 1. Thaw all reagents (Master Mix, primers, DNA template, nuclease-free water) and keep them on ice. | |
| 2. In a sterile 0.2 mL PCR tube, add 12.5 μL of 2x Master Mix. | |
| 3. Add 1 μL of Forward Primer (10 μM stock). | |
| 4. Add 1 μL of Reverse Primer (10 μM stock). | |
| 5. Add 2 μL of template DNA (concentration approx. 20 ng/μL). | |
| 6. Add 8.5 μL of nuclease-free water to reach a final volume of 25 μL. | |
| 7. Mix contents by gently flicking the tube, then perform a quick spin in a microcentrifuge to collect the liquid at the bottom. | |
| 8. Place the tube into the thermocycler. | |
| 9. Program the cycler or load pre-configured program: | |
| o Initial denaturation at 95°C for 3 minutes | |
| o 30 cycles of: 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min | |
| o Final extension at 72°C for 5 minutes | |
| 10. Start the program. | |