Source: Nocardia corallina

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℃), 200 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA, 50% glycerol.

Dilution buffer: EzDiluent B

10 mM Tris-HCl (pH 7.4 @ 25℃), 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA, 50% glycerol.

Heat Inactivation

EZ-CleanCut™ Nco I can be inactivated at 80℃ for 20 min.

Methylation sensitivity

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Not sensitive

Prolonged incubation

A minimum amount of enzyme required to digest 1-μg substrate DNA for 16 hr; 0.25 U.

Relative activity in EzBuffers

EzBuffer I: 50%

EzBuffer II: 100%

EzBuffer III: 10%

EzBuffer IV: 100%

FastCut Buffer: 100%

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●  Enzynomics_CR004_EZ-CleanCut™ Nco I_manual

● Material Safety Data Sheet

- Enzynomics_MSDS_CR004_EZ-CleanCut™ Nco I

● 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 hose 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℃ or 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 digested 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.