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R-loop formation measurement using the 2-AP-labeled DNA duplex
| Syringe A | Stock concentration | Final concentration | Volume (𝜇L) |
|---|---|---|---|
| DEPC-H2O | – | – | 180 |
| Cleavage Buffer | 5X | 1X | 60 |
| SpyCas9 | 10 𝜇M | 1 𝜇M | 30 |
| Guild RNA | 10 𝜇M | 1 𝜇M | 30 |
| Total volume | 300 | ||
| Incubate for 10 min at room temperature. | |||
| Syringe B | Stock concentration | Final concentration | Volume (𝜇L) |
| DEPC-H2O | – | – | 234, 228, 222, 210 |
| Cleavage Buffer | 5X | 1X | 60 |
| Duplex substrate2-AP | 10 𝜇M | 0.2, 0.4, 0.6, 1 𝜇M | 6, 12, 18, 30 |
| Total volume | 300 | ||
