FAQ: Why am I observing incomplete digestion with EnGen® Spy Cas9 Nickase?

Incomplete digestion may be due to the following factors:
  • Incorrect ratio of EnGen® Spy Cas9 Nickase to guide RNA and/or target site. For complete digestion we recommend a 10:10:1 or higher molar ratio of EnGen® Spy Cas9 Nickase : 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.
  • Use of a suboptimal buffer. Please use NEBuffer 3.1, which is included with the enzyme.