Incomplete digestion may be due to the following factors:
- Incorrect ratio of EnGen Lba Cas12a (Cpf1) to gRNA and/or target site. For complete digestion we recommend a 10:10:1 or higher molar ratio of EnGen Lba Cas12a (Cpf1) : gRNA : target site.
- Suboptimal sequence of the gRNA. Verify the sequence and design of the gRNA.
- Poor quality gRNA. Verify the integrity of the gRNA by gel electrophoresis.
- Use of a suboptimal buffer. Please use EnGen Lba Cas12a Diluent, which is included with the enzyme.
- When diluting 100 µM EnGen Lba Cas12a (Cpf1) for use in in vitro cleavage assays, supplementing reactions with 10 mM DTT can in some cases improve substrate cleavage.