(Streptomyces tubercidicus)
5’-A G G C C T-3’
3’-T C C G G A-5’
Reaction Temperature: 37°C
Prototype: StuI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Medium Buffer: 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, 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 unmethylated 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, 100 µg/ml bovine serum albumin, 1 µg of unmethylated lambda DNA. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.6 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 Endonucleases: Incubation of 40 units of StuI with 1 µg unmethylated 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.
3’-Exonuclease: 4, 8 and 16 units of StuI and 0.16 µg (1.23 pmol 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 -0.12-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 4, 8 and 16 units of StuI with 0.03 µg (0.37 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 37°C resulted in -0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.