| Component | Amount |
| 10× PfuTurbo Cx reaction buffer | 5 μL |
| dNTP Mix (10 mM each dNTP) | 1 μL |
| Self-designed primer-F (10 μM) | 1 μL (see Note 27) |
| Self-designed primer-R (10 μM) | 1 μL |
| Bisulfite-treated DNA (from previous step) | 1–2 μL |
| PfuTurbo Cx Hotstart DNA polymerase (2.5 U/μL) | 1 μL |
| Milli-Q water | To a final volume of 50 μL |
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