Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.Sticky ends from different Bpu10I sites may not be compatible.
Prolonged incubation with NdeI may lead to removal of additional nucleotides.
Sticky ends from different BsaI sites may not be compatible.BsaI can be used between 37°C and 50°C.
The 1-base overhangs produced by AhdI may be hard to ligate. Sticky ends from different AhdI sites may not be compatible.