Sticky ends from different BbsI sites may not be compatible.BbsI gradually loses activity when stored at -20°C.
The 1-base overhangs produced by PflFI may be hard to ligate.Sticky ends from different PflFI sites may not be compatible.
The 1-base overhangs produced by Tth111I may be hard to ligate.Sticky ends from different Tth111I sites may not be compatible.
Sticky ends from different BseRI sites may not be compatible.BseRI quickly loses activity at 37°C.Prolonged incubation with BseRI may lead to degradation of the DNA.
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
Efficient cleavage requires at least two copies of the SacII recognition sequence.
After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
BsaHI is typically used at 37°C, but is even more active at 60°C.