| Component | Amount/reaction (μl) |
| Linearized plasmid (CasRx pre-sgRNA cloning backbone) | 50 ng (depends on DNA concentration) |
| Annealed oligonucleotides | 1 |
| T4 DNA ligase T4 DNA ligase buffer (10 × ) | 1 (20 U) 2 |
| Nuclease-free water | Add up to 20 |
| Final volume | 20 |
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