Afe I

Enzyme nameAfe I
PrototypeEco47III
Known analogsAor51HI, Eco47III, FunI
SKUSE-E213
Turbo versionAvailable
High-concentration versionAvailable
Restriction typeType IIPiType IIP enzymes are classical Type II restriction enzymes with palindromic recognition sequences and symmetric cleavage sites.
Recognition site
5'… AGC▼GCT …3'
3'… TCG▲CGA …5'
SourceAn E.coli strain that carries the cloned Afe I gene from Alcaligenes faecalis T2774
Optimal bufferSE-buffer Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Optimal temperature37 °C
Inactivation temperature65 °C
Buffer activity
BGOWYROSE
10257575100100
Unit definitionOne 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 onLambda DNA (BamHI-digest)
Storage conditions10 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.
LigationAfter 10-fold overdigestion with enzyme more than 80% of DNA pBR322 fragments can be ligated and recut.
Nonspecific hydrolysisNo nonspecific activity was detected after incubation of 1 μg of DNA with 40 u.a. of enzyme for 16 hours at 37°C.
Methylation sensitivitynot tested
Reagents supplied with enzyme10 X SE-buffer Y
NotesThe 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
  1. Unpublished data Abdurashitov, M.A., Kileva, E.V., Shevchenko, A.V., Degtyarev, S.Kh Unpublished observations. (1994)
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