| Component | Amount per reaction (μL) | Final concentration |
|---|---|---|
| Rapid Ligase Buffer, 2X | 25 | 1X |
| BSA, 20 mg/mL | 0.25 | 0.1 mg/mL |
| FastDigest Esp3I, 1 unit/μL | 1 | 0.02 units/μL |
| T7 Ligase, 3 × 106 units/mL | 0.25 | 15 × 103 units/mL |
| sgRNA library insert from Step 8 | 15 ng | 0.3 ng/μL |
| Digested library plasmid backbone from Step 13 | 100 ng | 2 ng/μL |
| UltraPure water | up to 50 μL | |
| Total | 50 |
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