AluI
(Arthrobacter luteus)
5’- A G C T- 3’
3’- T C G A- 5’
Reaction Temperature: 37°C
Prototype: AluI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Acet Buffer: 20 mM Tris-acetate (pH 7.5 at 37°C), 10 mM magnesium acetate, 50 mM potassium acetate, 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 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, 1 µg of lambda DNA and 100 µg/ml bovine serum albumin. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 22°C), 100 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 500 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 AluI with 1 µg of 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 AluI and 0.08 µg (0.76 pmol of 3'-ends) of lambda/TaqI fragments (3'-labeled with T4 DNA Polymerase and [α³³P]dGTP and [α-³³P]dCTP), incubated for 1 hr at 37°C resulted in 0.07 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 AluI with 0.15 µg (1.27 pmol of 5’-ends) of [5’-³³P] lambda/HaeIII fragments for 1 hr at 37°C resulted in a less than –0.03 slope of %-end label released per unit of enzyme. Reaction volume 10 µl.