1. Rapid: the reaction can be completed in 5-15 min.
2. 37 ℃ is the most suitable reaction temperature.
3. Affected by EcoBl methylation, sequences may overlap and digestion blocking.
4. Enzyme inactivation after 20 minutes of incubation at 80 ℃.
5. 3-hour incubation did not show star activity, and delayed enzyme digestion may show star activity.