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Aat II

No Product Name Cat.# Conc Size Price(₩) Add
1 Aat II R026S 10 units/µl 500 units ₩ 80,000 추가
2 Aat II R026M 10 units/µl 1,000 units ₩ 144,000 추가
3 Aat II R026L 10 units/µl 2,500 units ₩ 320,000 추가
4 Aat II R026H 50 units/µl 2,500 units ₩ 320,000 추가
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Recognition & cleavage sequence


Source: Acetobacter aceti

 

Reaction conditions

1X EzBuffer IV, 37

1X FastCut Buffer, 37

 

FastCut Buffer

Enzynomics restriction enzyme can cut substrate DNA in 5-15 min with FastCut Buffer

 

1X EzBuffer IV

20 mM Tris-acetate (pH 7.9 @ 25), 50 mM potassium acetate, 10 mM magnesium acetate, 100 μg/ml BSA

 

Unit definition

One unit is defined as the amount of enzyme required for complete digestion of 1-μg bacteriophage λ at 37 for 1 hr in 50-μl reaction mixtures.

 

Storage

10 mM Tris-HCl(pH 7.4 @ 25), 50 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 200 μg/ml BSA, 50% Glycerol.

 

Dilution buffer: EzDiluent A

10 mM Tris-HCl (pH 7.4 @ 25), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA, 50% glycerol.

 

Heat Inactivation

Aat II can be inactivated at 65 for 20 min.

 

Methylation sensitivity

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: sensitive

 

Prolonged incubation

A minimum amount of enzyme required to digest 1-μg substrate DNA for 16 hr; 0.13 U.


Relative activity in EzBuffers

EzBuffer I: 0%

EzBuffer II: 25%

EzBuffer III: 25%

EzBuffer IV: 100%

FastCut Buffer: 100%

 

Note

More (3-5 fold) enzyme is required to cleave supercoiled DNA than lambda DNA. Activity decreases if buffer pH is not between 7.5 and 8.0 at 25. Cleavage of mammalian genomic DNA is blocked by CpG methylation.

● Material Safety Data Sheet

- Enzynomics_MSDS_R026_Aat II

 

● Quality control

Overdigestion assay

DNA digested for 16 hr in 50-μl reaction with 100 U of enzyme resulted in same the DNA band patterns as those obtained with 1 U of enzyme for 1 hr.

 

Endonuclease assay

Less than 5% of 1-μg of ΦX174 RFI is converted to RFII when the DNA is incubated with 50 U of enzyme at 37 for 4hr in 50-μl reaction.

 

Ligation and recutting

More than 95% of DNA fragments (1 μg) digested with 50-fold excess enzyme can be ligated by T4 DNA ligase (400 U) at 16 for 16 hr. Of the ligation products, > 95% can be re-cut.

 

Blue/white screening

To test the integrity of DNA ends, a plasmid, pSKM2 containing a unique site in the lacZ alpha gene is digestesd with 10-fold excess enzyme, ligated, and introduced into DH5α. The transformed cells are plated on X-gal/IPTG/Amp plates. The number of blue and white colonies formed are measured. Blue colonies indicate that an intactness of the test gene is maintained during the cleavage/ligation process. In contrast, white colonies fail to do so. Fewer than 1% white colonies are formed with enzyme.

 

Extreme pure (EP)

No detectable degradation of 32P-end labeled single-stranded and double-stranded (5’-, 3’-overhang and blunt end) oligonucleotides occurred during incubation with 100 U of enzyme for 4 hr.

 

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