(Streptomyces phaeochromogenes)
5’-G C A T G C-3’
3’-C G T A C G-5’
Reaction Temperature: 37°C
Prototype: SphI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Medium Buffer: 10 mM Tris-HCl (pH 7.5 at 37°C), 1 mM dithiothreitol, 10 mM MgCl2, 50 mM NaCl, 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: 10 mM Tris-HCl (pH 7.5 at 37°C), 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol and 1 µg of lambda 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, 5 mM β-mercaptoethanol, 100 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specyfic Endonucleases: Incubation of 40 units of SphI with 1 µg of lambda DNA at 37°C for 5 hrs (a 200-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
Nicking Activity (phiX-174): Incubation of 20 units of SphI with 1 µg of phiX-174 DNA at 37°C for 5 hrs resulted in <5% 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 SphI and 0.25 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 0.02-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 SphI 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.