HpaI
(Haemophilus parainfluenzae)
5’-G T T A A C-3’
3’-C A A T T G-5’
Reaction Temperature: 37°C
Prototype: HpaI
Inactivation Temperature (20 min): no
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: 20 mM Tris-acetate (pH 7.5 at 37°C), 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 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 7.5 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 Endonucleases: Incubation of 10 units of HpaI 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: 6, 12 and 24 units of HpaI and 0.11 µg (0.80 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 37°C resulted in 0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 6, 12 and 24 units of HpaI with 0.016 µg (0.15 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.
Nicking Activity (pBR322): Incubation of 20 units of HpaI with 1 µg of pBR322 at 37°C for 5 hrs resulted in <5% conversion from Form I to II and <1% conversion from Form II to III as determined by agarose gel electrophoresis.