(Neisseria denitrificans)
5'-C A T A T G -3'
3'-G T A T A C -5'
Reaction Temperature: 37°C
Prototype: NdeI
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), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin and 1 µg lambda DNA. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 50 mM Tris-HCl (pH 7.5 at 22°C), 0.1 mM EDTA, 1 mM dithiothreitol, 500 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 20, 35 and 50 units of NdeI with 1 µg of lambda DNA at 37°C for 10 hrs (a 250-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
3'-Exonuclease: 5, 10 and 20 units of NdeI and 0.19 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 0.007 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.
5'-Exonuclease/5'-Phosphatase: Incubation of 5, 10 and 20 units of NdeI with 0.25 µg of 5'-ends of [5'-³²P] lambda/HaeIII fragments for 1 hr at 37°C resulted in 0.000 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.