(Pseudomonas inaequalis)
5’-A C C G G T-3’
3’-T G G C C A-5’
Reaction Temperature: 37°C
Prototype: Age I
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x PinAI Buffer: 20 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 50 mM KCl, 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-HCl (pH 7.4), 5 mM MgCl2, 50 mM KCl, 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: 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 100 mM NaCl, 10 mM ß-mercaptoethanol, 0.05% (v/v) Triton X-100, and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Endonuclease: Incubation of 10, 20, and 30 units of PinAI with 1 µg of lambda DNA at 37°C for 5 hr (a 150-fold over-digestion) resulted in the same sharp characteristic banding pattern as 4 U/µg in the standard 1hr assay reaction, as determined by agarose gel electrophoresis. Reaction volume 50 µl.
Nickase: Incubation of 10, 20, and 30 units of PinAI with 1 µg of ØX-174 DNA at 37°C for 5 hr (a 150-fold over-digestion) resulted in ≤40% conversion of RF I to RF II and no conversion to RF III, as determined by agarose gel electrophoresis. Reaction volume 50 µl.
3'-Exonuclease: Incubation of 2, 5, 10, and 20 units of PinAI and 5 pmoles of 3'-ends of lambda/TaqI fragments (3'-labelled with Klenow exo- and [3H]dCTP), incubated for 1 hr at 37°C resulted in ≤0.3 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.
5'-Exonuclease/5'-Phosphatase: Incubation of 2, 5, 10, and 20 units of PinAI with 0.25 µg 5'-ends of [5'-32P]lambda/HaeIII fragments for 1 hr at 37°C resulted in ≤0.3 slope of %-end label released per unit of enzyme. Reaction volume 50 µl.
Ligation: Incubation of 2 units of PinAI with 1 µg of lambda DNA at 37°C produces fragments that ligate with ≥95% efficiency. The recut efficiency of these ligated fragments is 100%.