(Thermus thermophilus 111)
5’-G A C N N N G T C-3’
3’-C T G N N N C A G-5’
Reaction Temperature: 65°C
Prototype: Tth111I
Inactivation Temperature (20 min): no
Reaction Buffer:
1x Low Buffer: 10 mM Tris-HCl (pH 7.0 at 37°C), 10 mM MgCl2, 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 Ad-2 DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 10 mM Tris-HCl (pH 7.0 at 37°C), 10 mM MgCl2, 1 mM dithiothreitol, 1 µg of Ad-2 DNA and 100 µg/ml bovine serum albumin. Incubation is at 65°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 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 Endonucleases: Incubation of 10 units of Tth111I with 1 µg Ad-2 DNA at 65°C for 15 hrs under oil (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 Tth111I 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 65°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 Tth111I with 0.05 µg of 5’-ends of [5’-³²P] lambda/HaeIII fragments for 1 hr at 65°C resulted in 0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.