(Nocardia otitidis-caviarum)
5’-G C G G C C G C-3’
3’-C G C C G G C G-5’
Reaction Temperature: 37°C
Prototype: NotI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x High Buffer: 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 1 mM ditiothreitol, 100 µg/ml bovine serum albumin.
Unit Definition: One unit is the amount of enzyme required to completely digest 1 µg of Ad-2 DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 50 mM Tris-HCl (pH 7.5 at 37°C), 100 mM NaCl, 10 mM MgCl2 and 1 µg of Ad-2 DNA. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 20 mM Tris-HCl (pH 7.5 at 22°C), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01% (v/v) Triton™X-100, 500 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 10 units of NotI with 1 µg of Ad-2 DNA at 37°C for 16 hrs (a 160-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
Nicking Activity (SV40): Incubation of 5, 10, and 20 units of NotI with 1 µg of SV40 DNA at 37°C for 5 hrs resulted in <10% conversion from Form I to II and 0% conversion from Form II to III as determined by agarose gel electrophoresis.
Nicking Activity (FX-174): Incubation of 5, 10, and 20 units of NotI with 1.0 µg of phiX-174 DNA at 37°C for 5 hrs resulted in <10% conversion from Form I to Form II and 0% conversion from Form II to Form III as determined by agarose gel electrophoresis.
3'-Exonuclease: 5, 10 and 20 units of NotI and 1.0 pmoles of 3'-ends of lambda/TaqI fragments (3'-labeled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 37°C resulted in 0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5'-Exonuclease/5'-Phosphatase: Incubation of 5, 10 and 20 units of NotI with 0.05 µg of 5'-ends of [5'-³²P]lambda/HaeIII fragments for 1 hr at 37°C resulted in -0.05-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.