A mismatch-sensitive T7 endonuclease 1 test (New England Biolabs) was used to confirm that DNA cleavage and targeted sequence disruption occurred at the specified spot. DNA was extracted from the pSpcas9-sgRNA1/2 group,
pSpcas9-group, and blank group cells using the Favor PrepTM GEL Purification and DNA extraction kit (FAVORGEN Biotech Corp-Taiwan, according to the manufacturer’s instructions). In three separate microtubes, 10 μL (200 ng) of each DNA sample was combined with 2 μL of 10X NE-Buffer 2 buffer and 19 μL of nuclease-free water. At 95 °C for 10 minutes, the samples were heated. Then, it was allowed to cool at room temperature gradually.
Nineteen microliters of each sample were combined with 1 μL of T7 endonuclease I (5units.μL-1) and incubated at 37 °C for 15 minutes before being examined on an agarose gel.
Band intensities were measured using Tanon-electrophoretic software (Tanon Science & Technology Co., Ltd., Shanghai, China), and the targeted disruption was seen as described by Zhen Shuai ( 26 (link)
).
pSpcas9-group, and blank group cells using the Favor PrepTM GEL Purification and DNA extraction kit (FAVORGEN Biotech Corp-Taiwan, according to the manufacturer’s instructions). In three separate microtubes, 10 μL (200 ng) of each DNA sample was combined with 2 μL of 10X NE-Buffer 2 buffer and 19 μL of nuclease-free water. At 95 °C for 10 minutes, the samples were heated. Then, it was allowed to cool at room temperature gradually.
Nineteen microliters of each sample were combined with 1 μL of T7 endonuclease I (5units.μL-1) and incubated at 37 °C for 15 minutes before being examined on an agarose gel.
Band intensities were measured using Tanon-electrophoretic software (Tanon Science & Technology Co., Ltd., Shanghai, China), and the targeted disruption was seen as described by Zhen Shuai ( 26 (link)
).
