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EZ-CleanCut™ Nco I
EZ-CleanCut™ Nco I with high purity and fidelity has greatly reduced star activity than Nco I.
Recognition & cleavage sequence
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%
"Shipping
only to Asia."
● 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.