(Rhodopseudomonas sphaeroides)
5’-G T A C-3’
3’-C A T G-5’
Reaction Temperature: 37°C
Prototype: RsaI
Inactivation Temperature (20 min): 65°C
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 lambda 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, 100 µg/ml bovine serum albumin and 1 µg of lambda DNA. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.4 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-specyfic Endonucleases: Incubation of 10 units of RsaI with 1 µg lambda DNA at 37°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 RsaI and 0.21 µg (1.61 pmol of 3’-ends) of lambda/TaqI fragments (3’-labeled with T4 DNA Polymerase and [°H]dGTP and [³H]dCTP), incubated for 1 hr at 37°C resulted in 0.04-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 RsaI with 0.04 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.