| Component | Amount (μl) | Final concentration |
|---|---|---|
| Herculase II PCR buffer, 5× | 10 | 1× |
| dNTP, 100 mM (25 mM each) | 0.5 | 1 mM |
| U6 PCR template (pSpCas9(BB)) | 1 | 0.2 ng μl−1 |
| U6-Fwd primer (universal) | 1 | 0.2 μM |
| U6-Rev primer (sgRNA specific) | 1 | 0.2 μM |
| Herculase II fusion polymerase | 0.5 | |
| Distilled water | 36 | |
| Total | 50 |
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