Unit substrate: Lambda DNA (HindIII digest).

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25°C), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37^oC.

Absence of contaminants: 80 units of NheI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/Hind III digest at 37^oC. After 100-fold overdigestion with NheI, greater than 98% of the DNA fragments can be ligated and recut.

Storage buffer: 200 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 0.15% Triton X-100, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65°C for 20 minutes..

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

Note: Activity inhibited by salt concentrations >100mM. Cleaves to leave a 5’ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI, or XbaI.

Star activity: Low salt, high glycerol (>5%) concentrations, pH >8.0 or large excess of the enzyme may result in star activity.   

Reagents supplied: 10x A and 10x K buffer