Afe I

Description

Recognition site and hydrolysis position:

Source: An E.coli strain that carries the cloned Afe I gene from Alcaligenes faecalis T2774
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.
Assayed on:
Lambda DNA (BamHI-digest)
Optimal SE-buffer: SE-buffer Y
Enzyme activity (%):

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:
No nonspecific activity was detected after incubation of 1 μg of DNA with 40 u.a. of enzyme for 16 hours at 37°C. Reagents Supplied with Enzyme 10 X SE-buffer Y
Reagents Supplied with Enzyme:
Methylation sensitivity: not tested
Notes: 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.
References:
Abdurashitov, M.A., Kileva, E.V., Shevchenko, A.V., Degtyarev, S.Kh. Unpublished observations. (1994)