PCR approach in 20 μL with hot-start (qualitative PCR)
Procedure
As usually more than one PCR sample is run simultaneously, reaction and enzyme mix are prepared separately. The enzyme mix is added to the reaction mix after starting the PCR and after pre-heating the reaction mix to 94 ºC (hot start). A typical 50 μL approach that yields enough material for later cloning procedures is made up as follows:
| ☐ | H2O | 12 µL |
| ☐ | 10x Taq/Pfu buffer | 1.5 μL |
| ☐ | Primer 1 | 0.4 μL (10 pmol) |
| ☐ | Primer 2 | 0.4 μL (10 pmol) |
| ☐ | Template | 0.2 μL (~10 ng) |
| ☐ | dNTPs | 0.2 μL |
| ☐ | Total | 15 μL |
| ☐ | H2O | 4 µL |
| ☐ | 10x Taq/Pfu buffer | 0.5 μL |
| ☐ | Taq Polymerase | 0.5 μL |
| ☐ | – | – |
| ☐ | – | – |
| ☐ | – | – |
| ☐ | Total | 5 μL |
Touch-down PCR:
1 Hot start 94 ºC -> Pause (addition of the enzyme mix)
7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down)
2 Denaturation 94 ºC 20 seconds
3 Hybridization 64-58 ºC 20 seconds
4 Elongation 72 ºC depending on fragment size (1 min/750 bp)
30 repetitions of steps 2-4 with hybridization temperature of 58 ºC
5 Elongation 72 °C 5-10 min
6 Stop 8°C
(An alternative to touch-down PCR is a gradient PCR, with a gradient of annealing temperatures usually between 50 – 64°C)