Linearized vectors were incubated with the indicated enzymes (10 units/µg) for 60 minutes at the recommended incubation temperature and NEBuffer for each enzyme. Following ligation and transformation, cleavage efficiencies were determined by dividing the number of transformants from the digestion reaction by the number obtained from religation of the linearized DNA (typically 100–500 colonies) and subtracting from 100%. “Base Pairs from End” refers to the number of double-stranded base pairs between the end of the recognition site and the terminus of the fragment; this number does not include the single-stranded overhang from the initial cut. Since it has not been demonstrated whether these single-stranded nucleotides contribute to cleavage efficiency, 4 bases should be added to the indicated numbers when designing PCR primers. Average efficiencies were rounded to the nearest whole number; experimental variation was typically within 10%.

Note: This data represents the minimum number of bases that will work, but is not recommended for maximum cleavage. As a general rule, enzymes not listed below require 6 bases pairs on either side of their recognition site to cleave efficiently.