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. Author manuscript; available in PMC: 2021 Aug 14.
Published in final edited form as: Methods Enzymol. 2019 Jan 14;616:289–311. doi: 10.1016/bs.mie.2018.10.022

Table 3.

DNA off rate measurement assay using the γ32P-labeled TS DNA duplex

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.