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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » Acc65I (KpnI*)

Acc65I (KpnI*)

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Specifications

5'-G^G T A C C-3' 3'-C C A T G^G-5'
Unlike KpnI, Acc65I produces DNA fragments with a 4-base 5'-extension.
Catalog #A0200
Concentration 10 units/ul
Source: Acinetobacter aceti 655
Recommended ConditionsStorage Buffer: 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM sodium chloride, 0.1mg/ml BSA. Incubate at 37°C.
Diluent Buffer 10mM Tris-HCl, pH 7.4 at 25°C, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
Storage Buffer 10mM Tris-HCl, pH 7.4 at 25°C, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
Quality Control
Overdigestion Assay
No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Acc65I.
Ligation/Recutting Assay
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Acc65I approximately 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.01uM. More than 90% of these can be recut.
Labeled Oligonucleotide (LO) Assay (endonuclease, exonuclease and phosphatase)
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
pUC57 was digested at a unique site with 10 units of enzyme for 16 hours. After religation and transformation 0.3% of white colonies were detected.
Methylation Effects
Acc65I cleaves the hemi-methylated sequence GGTACm5C. Cleavage is blocked when both strands are methylated at positions
5' GGTACm5C3' 3'm5CCATG G5'
[Overlapping Dcm or CG methylation may influence DNA cleavage]
Stability during Prolonged Incubation
A minimum of 0.3 units of enzyme are 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.
Compatible EndsBshNI, Bsp1407I, Pfl23II, TatI
Number of Recognition Sites in DNA
Lambda 2
PhiX174 0
M13mp18/19 1
pBR322 0
pUC18/19 1
pUC57 1
pTZ19R/U 1
pBluescriptIIKS(-/+) 1
pBluescriptIISK(-/+) 1
pACYC177 0
pACYC184 0
Unit Definition
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA-BamHI fragments in 1 hour at 37°C in 50ul of assay buffer.


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