| Component | 50 μL | Final Concentration | |
|---|---|---|---|
| 1 | Nuclease–free water | 33 | |
| 2 | 5X Phusion HF buffer | 10 | 1X |
| 3 | 10 mM dNTPs | 1 | 200 μM |
| 4 | 10 μM Cas9_forward | 2.5 | 0.5 μM |
| 5 | 10 μM Cas9_reverse | 2.5 | 0.5 μM |
| 6 | pX330 (50 ng/μL) | 0.5 | <250 ng |
| 7 | Phusion DNA Polymerase | 0.5 | 1.0 units/50 μL |
| total | 50 |
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