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