AclW I

Enzyme name AclW I
Prototype BinI
Known analogs AlwI, BinI, BspPI
SKU SE-E211
Turbo version Not available
High-concentration version Not available
Restriction type Type IISiType IIS enzymes recognize asymmetric DNA sequences and cleave outside the recognition site at a defined distance. They are widely used in Golden Gate assembly.
Recognition site
5'… GGATC(N)4▼ …3'
3'… CCTAG(N)5▲ …5'
Source Acinetobacter calcoaceticus W2131
Optimal buffer SE-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
Inactivation temperature 65 °C
Buffer activity
BGOWYROSE
75500010030
Unit definition One unit of the enzyme is the amount required to hydrolyze 1 μg of Lambda DNA (dam-) in 1 hour at 37°C in a total reaction volume of 50 μl.
Assayed on Lambda DNA (dam-)
Storage conditions 10 mM Tris-HCl (pH 7.5); 100 mM KCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA; 50% glycerol; Store at -20°C.
Ligation After 3-fold overdigestion with enzyme about 50% of the DNA fragments can be ligated with T4 DNA ligase and recut. In the presence of 10% PEG
Nonspecific hydrolysis No nonspecific activity was detected after incubation of 1 μg of Lambda DNA with 6 u.a. of enzyme for 16 hours at 37°C.
Methylation sensitivity Blocked by Dam methylation (G m ATC) G GATC
Reagents supplied with enzyme 10 X SE-buffer Y, BSA
Notes To obtain 100% activity, BSA should be added to the 1x reaction mix to a final concentration of 100 μg/ml. Do not use BSA for long incubation.
References
  1. Unpublished data Abdurashitov, M.A., Belichenko, O.A., Shevchenko, A.V., Degtyarev, S.Kh Unpublished observations. (1994)
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