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Nae I (Pdi I)
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.
● Material Safety Data Sheet
● 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.