2.6 Activity assay of rSaCas9
The vector containing the CCD8 promoter target sequence (0.5 µg
pJET_CCD8) was mixed with rSaCas9 (150 ng from EF#2), one of the ten
(T1-T10) in vitro transcribed sgRNAs (400–1300 ng) in reaction
buffer (NEBuffer1, 10 mM Bis-Tris-Propane-HCl, 10 mM
MgCl2, 1 mM DTT, pH 7). The reaction mixture (final
volume 30 µl) was incubated at 37 °C for one hour, and aliquots were
analyzed by agarose electrophoresis (0.8% (w/v) agarose in TBE, 120 V,
30 min).