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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

Figure 4.

Figure 4.

(A) The DNA dissociation rate measurement was conducted by preincubating radiolabeled DNA duplex and Cas9.gRNA for 30 min in the absence of Mg2+. Following by addition of 20X excess of cold trap DNA for varying incubation time points (0, 1, 5, 10 ,20, 30, 45, and 60 min), after adding Mg2+ to start the reaction for 30 sec. The scheme is shown above the gel image. (B) The plot of product formation as a function of time was fit by the single-exponential decay equation to define the DNA dissociation rate from Cas9.gRNA (koff,DNA = 0.0024 s−1). This figure is adapted from Gong et al. 2018.