The minimum number of units that resulted in complete digestion of 1 μg of substrate DNA in 16 hours is 0, 25. AfeI cleaves supercoiled and linear plasmid DNA (pBR322) at a roughly equal rate. AfeI cleaves Lambda DNA/BamHI digest at a rate 3-4 times higher than plasmid DNA.
Unit definition | One unit of the enzyme is the amount required to hydrolyze 1 μg of Lambda DNA (BamHI-digest) in 1 hour at 37°C in a total reaction volume of 50 μl. |
Recognition sites | AGC↑GCT |
Source | E.coli strain that carries the cloned Afe I gene from Alcaligenes faecalis T2774 |
Assayed on | Lambda DNA (BamHI-digest) |
Optimal buffer | Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.) |
Optimal temperature | 37°C |
Storage conditions | 10 mM Tris-HCl (pH 7.6); 50 mM NaCl; 0.1 mM EDTA; 1 mM DTT; 200 μg/ml BSA; 50% glycerol; Store at -20°C. |
Ligations | After 10-fold overdigestion with enzyme more than 80% of DNA pBR322 fragments can be ligated and recut. |
Non-specific hydrolisis | fragments can be ligated and recut.|Non-specific hydrolisis No nonspecific activity was detected after incubation of 1 μg of DNA with 40 u.a. of enzyme for 16 hours at 37°C. |
Methlation effect | Methylation sensitivity not tested |
Thermal inactivation | enzyme is inactivated by incubation at 65°C for 20 mins. |
Campatible Ends | Aor51HI , Eco47III, FunI |
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