| Component | Amount per reaction (μL) | Final concentration |
|---|---|---|
| Q5 Reaction Buffer (5X) | 5 | 1X |
| dNTPs (10 mM) | 0.5 | 200 μM |
| Forward Primer mix from Step 26 (10 μM) | 1.25 | 0.5 μM |
| SPCR_MiSeq3_rev (Table 1) (10 μM) | 1.25 | 0.5 μM |
| Template DNA from Step 24 | 0.8 | - |
| Q5 High-Fidelity DNA Polymerase | 0.25 | - |
| SYBR Green I from Step 25 (5X) | 5 | 1X |
| H2O | 10.95 | - |
| Total | 25 | - |
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