Recognition & cleavage sequence

Source: Haemophilus parahaemolyticus

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 (dam^-)

at 37℃ for 1 hr in 50-μl reaction mixtures.

Storage

10 mM Tris-HCl (pH 7.5 @ 25℃), 300mM 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

Hph I can be inactivated at 65℃ for 20 min.

Methylation sensitivity

dam methylation: Conditionally 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: 100%

EzBuffer II: 75%

EzBuffer III: 10%

EzBuffer IV: 100%

FastCut Buffer: 100%

Note

Activity is inhibited by dam methylation partially overlapping its recognition sequence.

●  Enzynomics_R053_Hph I_manual

● Material Safety Data Sheet

- Enzynomics_MSDS_R053_Hph I

● Quality control

Overdigestion assay

DNA digested for 16 hr in 50-μl reaction with 100U of enzyme resulted in same the DNA band patterns as those obtained with 1 U of enzyme fo1 hr.

Ligation and recutting

More than 95% of DNA fragments (1 μg) digestewith 50-fold excess enzyme can be ligated by T4DNA 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.