BssHII
(Bacillus stearothermophilus H3)
5’-G C G C G C-3’
3’-C G C G C G-5’
Reaction Temperature: 50°C
Prototype: BsePI
Inactivation Temperature (20 min): 80°C
Reaction Buffer:
1x High Buffer: 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 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 lambda DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 50 mM Tris-HCl (pH 7.5 at 37°C), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin and 1 µg of lambda DNA. 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, 100 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 20 units of BssHII with 1 µg of lambda DNA at 50°C for 5 hrs (a 100-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
Nicking Activity (SV40/pBR322): Incubation of 5, 10, and 20 units BssHII with 1 μg of SV40/pBR322 DNA at 50°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 BssHII and 0.17 µg (1.30 pmol of 3’-ends) of lambda/TaqI fragments (3’-labeled with T4 DNA Polymerase and [³³P]dGTP and labeled [³³P]dCTP), incubated for 1 hr at 50°C resulted in -0.005 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 BssHII with 0.05 µg of [5’-³³P] lambda/HaeIII fragments for 1 hr at 50°C resulted in a less than 0.01 slope of %-end label released per unit of enzyme. Reaction volume 10 µl.