FAQ: Why do I observe incomplete digestion/editing?

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.