FAQ: Will DNase I work in NEB buffers 1-4?

Yes, but not as efficiently. DNase I is a Calcium dependent enzyme so it works best in it’s own buffer which contains Calcium Chloride. It is recommended that even if you are using NEB buffers 1-4 that you also add DNase I buffer or .5mM CaCl2 to the reaction. Also, DNase I does not like high salt, as contained in NEB buffer 3. It works least efficiently in buffer 3, but will still give 50-75% digestion.