5'-^ 7(N) G A A C (N)6 T C C (N)12–13^-3' 3'-^12–13(N) C T T G (N)6 A G G (N)7 ^-5'
Source: E.coli that carries the cloned aloIR gene from Acinetobacter lwoffi Ks4–8
Buffer: 10mM Tris-HCl (pH 8.5), 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 30°C. Incubation at 37°C results in 20% activity.
Diluent Buffer: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. For longer periods–the Storage Buffer should be used.
Storage Buffer: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
Overdigestion Assay
No detectable change in the specific fragmentation pattern is observed after 10-fold overdigestion (5u/ug lambda DNA x 2 hours) with AloI (see Star Activity).
Ligation/Recutting Assay
After 10-fold overdigestion (0.6u/ug DNA x 17 hours) with AloI, more than 70% of the DNA fragments can be ligated at a 5'-termini concentration of 0.06uM. More than 80% of these can be recut.
Labeled Oligonucleotide (LO) Assay
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of restriction endonuclease for 4 hours.
Blue/White Cloning Assay
The mix of pUC57/HindIII, pUC/PstI and pUC57/Eco32I digests was incubated with 10units of enzyme for 16 hours. After religation and transformation 0.6% of white colonies were detected.
Star Activity
An excess of enzyme (6u/ug lambda DNA x 2 hours) may result in star activity.
Methylation Effects
AloI does not cut GGA(N)6GTTm5C. Overlapping CG methylation may influence DNA cleavage.
Stability during Prolonged Incubation
A minimum of 0.1units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal Inactivation
Enzyme is inactivated by incubation at 65°C for 20min.
Number of Recognition Sites in DNA
Note
AloI produces double-strand cuts on both sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3' side of the recognition sequence (12 or 13 nt away). Addition of SAM to the reaction mixture results in incomplete cleavage.
Unit Definition
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 30°C in 50ul of assay buffer.