Recognition & cleavage sequence

Source: Nocardia aerocolonigenes

Isoschizomer : Pdi I

Reaction conditions

1X EzBuffer I, 37℃

1X FastCut Buffer, 37℃

FastCut Buffer

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

1X EzBuffer I

10 mM Bis Tris propane-HCl (pH 7.0 @ 25℃), 10 mM MgCl₂, 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 NaCl, 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

Nae I 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; 0.5 U.

Relative activity in EzBuffers

EzBuffer I: 100%

EzBuffer II: 100%

EzBuffer III: 25%

EzBuffer IV: 100%

FastCut Buffer: 100%

Note

It is an isoschizomer of NgoM lV. Cleavage of mammalian genomic DNA is blocked by CpG methylation. It displays marked site preference, while NgoM IV has less site preference. Two recognition sequences are required for cleavage. One of the two acts as an effector site. It is sensitively affected by the locations of the recognition sequence. For example, if the two sites are too close, Nae I is not efficient in cleaving one of the two.

●  Enzynomics_R035_Nae I_manual

● Material Safety Data Sheet

- Enzynomics_MSDS_R035_Nae 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 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. 

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.