Ligation can also be performed in any of the standard restriction endonuclease NEBuffers, including rCutSmart® Buffer, or in T4 Polynucleotide Kinase Buffer if supplemented with 1 mM ATP. Please be sure to use riboATP (NEB #P0756) as deoxyriboATP will not work. When using NEBuffer 3 or r3.1, high levels of salt in the DNA preparation may decrease ligation efficiency. To see its % functional activity in rCutsmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.