Incomplete digestion may be due to the following factors:
- Suboptimal ratio of Cas9 Nuclease to guide RNA and target site. For complete digestion, we recommend a 10:10:1 or higher molar ratio of Cas9 Nuclease: guide RNA: DNA target. For plasmid linearization, higher ratios of nuclease/sgRNA may be required.
- Suboptimal sequence of the guide RNA. Verify the sequence and design of the guide RNA.
- Poor quality guide RNA. Verify the integrity of the guide RNA by gel electrophoresis.
- Suboptimal buffer. Please use the NEBuffer™ r3.1 included with the enzyme.
