(Proteus vulgaris)
5’-C A G C T G-3’
3’-G T C G A C-5’
Reaction Temperature: 37°C
Prototype: PvuII
Inactivation Temperature (20 min): no
Reaction Buffer:
1x Medium Buffer: 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCl, 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), 50 mM NaCl, 10 mM MgCl2, 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.
To avoid star activity it is not recommended:
- to use more than 15 units per reaction;
- to incubate over 1 hr.
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 Endonuclease: Incubation of 30 units of PvuII with 1 µg of lambda DNA at 37°C for 16 hrs (a 480-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 PvuII and 0.15 µg (1.15 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 -0.03-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 PvuII with 0.20 µg (1.8 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.