AcvI
(Aeromonas caviae)
5'-C A C G T G- 3'
3'-G T G C A C- 5'
Reaction Temperature: 37°C
Prototype: PmaCI
Inactivation Temperature (20 min): 80°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 Ad-2 DNA in 1 hr. Total reaction volume is 50 µl.
Notes:
It is not recommended:
- to perform digestion for over 4 hours;
- to use more than 10 units of enzyme per 1 µg of DNA.
These conditions may result in star activity.
Assay Conditions: 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 and 1 µg of Ad-2 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), 0.1 mM dithiothreitol, 50 mM KCl, 10 mM MgCl2, 200 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 AcvI with 1 µg of Ad-2 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 AcvI and 0.13 µg 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 0.06-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.