NotI is a restriction enzyme purified from Nocardia otitidis-caviarum.

Unit substrate: Adenovirus-2 DNA.

Unit calculation assay conditions: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 5 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37°C.

Absence of contaminants: 80 units of Not I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Adeno-2 DNA at 37°C. After 30-fold overdigestion with Not I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Storage buffer: 500 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100, 500 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: 65^oC for 20 minutes.

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked

Note: Supercoiled plasmids may require up to 5-fold more Not I for complete digestion than linear DNAs.

Reagents supplied: 10x U_NotI  buffer