FokI
(Flavobacterium okeanokoites)
5'-G G A T G (N)9-3'
3’-C C T A C (N)13–5’
Reaction Temperature: 37°C
Prototype: FokI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Medium Buffer: 10 mM Tris-HCl, (pH 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin.
It is not recommended to use more than 1 unit per 1 μg of DNA and incubate longer than 2 hours.
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), 10 mM MgCl2, 50 mM NaCl, 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 7.4 at 25°C), 50 mM NaCl, 0.1 mM EDTA, 0.1 % Tween 20, 1 mM dithiothreitol, 200 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 FokI with 1 µg of lambda DNA at 37°C for 16 hrs resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
3’-Exonuclease: 2, 5 and 10 units of FokI and 0.16 µg (1.23 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 0.05-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 2, 5 and 10 units of FokI with 0.03 µg,(0.24 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 37°C resulted in 0.03-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.