(Nocardia corallina)
5'-C C A T G G-3'
3'-G G T A C C-5'
Reaction Temperature: 37°C
Prototype: NcoI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Acet Buffer: 20 mM Tris-acetate (pH 7.5 at 37°C), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin.
Unit Definition: One unit is the amount of enzyme required to completely digest 1 µg of lambda DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 20 mM Tris-acetate (pH 7.5 at 37°C), 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 µg of lambda DNA and 100 µg/ml bovine serum albumin. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 22°C), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 60 units of NcoI with 1 µg of lambda DNA at 37°C for 5 hrs (a 300-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
Nicking Activity (pBR322) : Incubation of 30 units of NcoI with 1 µg of pBR322 at 37°C for 5 hrs resulted in <1% conversion from Form I to II and 0% conversion from Form II to III as determined by agarose gel electrophoresis.
3’-Exonuclease: 5, 10 and 20 units of NcoI and 0.13 µg 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 NcoI with 0.05 µg of 5’-ends of [5’-³²P]lambda/HaeIII fragments for 1 hr at 37°C resulted in -0.02-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.