HpaII
(Haemophilus parainfluenzae)
5'-C C G G-3'
3'-G G C C-5'
Reaction Temperature: 37°C
Prototype: HpaII
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, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin, 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 HpaII 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 HpaII and 0.15 µg (1.15 pmol of 3’-ends) of lambda/TaqI fragments (3’-labelled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 37°C resulted in 0.03-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 HpaII with 0.12 µg (1.07 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 37°C resulted in 0.04-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
Ligation-recut Assay: 7.5 µg of lambda DNA was incubated with 30 units of HpaII for 1 hr at 37°C in a reaction volume of 150 µl. 1.1 µg was removed for an assay. The remaining DNA was incubated with 2 Weiss units of T4 DNA Ligase for 1 hr at 22°C. 3.2 µg were removed for an assay. The remainder of the DNA in the ligation mixture was recovered and then incubated with 50 units of Hpa II for 1 hr at 37°C. Aliquots of the cut, ligated and recut DNA were electrophoresed on an agarose gel. Results show >95% ligation and >95% recutting.