FAQ: Why do I observe incomplete digestion?

Incomplete digestion may be due to the following factors:

  • Incorrect 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 : target site.
  • 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 3.1 included with the enzyme.