(Streptomyces fimbriatus)
5’-G G C C N N N N N G G C C-3’
3’-C C G G N N N N N C C G G-5’
Reaction Temperature: 50°C
Prototype: SfiI
Inactivation Temperature (20 min): no
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 complete 98% digestion of 1 µg of Ad-2 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, 1 µg of Ad-2 DNA and 100 µg/ml bovine serum albumin. Incubation is at 50°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, 200 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 SfiI with 1 µg of Ad-2 DNA at 50°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.
3'-Exonuclease: 5, 10 and 20 units of SfiI and 0.09 µg (0.65 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 50°C resulted in 0.03 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 SfiI with 0.08 µg (0.74 pmol of 5'-ends) of [5'-³²P] lambda/HaeIII fragments for 1 hr at 50°C resulted in a less than 0.04-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.