Table 3.
Syringe A | Stock concentration | Final concentration | Volume (𝜇L) |
---|---|---|---|
DEPC-H2O | – | – | 64 |
Cleavage Buffer | 5X | 1X | 20 |
SpyCas9 | 10 𝜇M | 400 nM | 4 |
Guild RNA | 10 𝜇M | 400 nM | 4 |
Duplex substrate | 0.5 𝜇M | 40 nM | 8 |
Total volume | 100 | ||
Incubation Cas9.gRNA for 10 min at room temperature, and then adding DNA to incubate Cas9.gRNA.DNA for 30 min at 37°C. | |||
Syringe B | Stock concentration | Final concentration | Volume (𝜇L) |
DEPC-H2O | – | – | 64 |
Cleavage Buffer | 5X | 1X | 20 |
20X cold trap DNA | 5 𝜇M | 800 nM | 16 |
Total volume | 100 | ||
Incubation at various time points (0–30 min) at 37°C. | |||
Syringe C | Stock concentration | Final concentration | Volume (𝜇L) |
DEPC-H2O | – | – | 120 |
Cleavage Buffer | 5X | 1X | 40 |
MgCl2 | 100 mM | 20 mM | 40 |
Total volume | 200 | ||
Allow the reaction to proceed for 30 sec. |