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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » AloI

AloI

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Specifications

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'
Catalog #A1356
Concentration 2u/ul
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 AssayNo 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 AssayAfter 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) AssayNo 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 AssayThe 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 ActivityAn excess of enzyme (6u/ug lambda DNA x 2 hours) may result in star activity.
Methylation EffectsAloI does not cut GGA(N)6GTTm5C. Overlapping CG methylation may influence DNA cleavage.
Stability during Prolonged IncubationA minimum of 0.1units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal InactivationEnzyme is inactivated by incubation at 65°C for 20min.
Number of Recognition Sites in DNA
Lambda7
PhiX1740
M13mp18/191
pBR3220
pUC18/190
pUC570
pTZ19R/U1
pBluescriptIIKS(-/+)1
pBluescriptIISK(-/+)1
pACYC1770
pACYC1840
NoteAloI 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 DefinitionOne 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.


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