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Afl II (BspT I)

No Product Name Cat.# Conc Size Price(₩) Add
1 Afl II R042S 4 units/µl 2,000 units ₩ 85,000 추가
2 Afl II R042M 4 units/µl 4,000 units ₩ 153,000 추가
3 Afl II R042L 4 units/µl 10,000 units ₩ 340,000 추가
4 Afl II R042H 20 units/µl 10.000 units ₩ 340,000 추가
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Recognition & cleavage sequence


Source: Anabaena flos-aquae


IsoschizomerBfr I , BspT I, Bst98 I, BstAF I, MspC I, Vha464 I

 

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), 50 mM KCI, 0.5 mM EDTA, 1 mM dithiothreitol, 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

Afl II can be inactivated at 65 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; 0.13 U.

 

Relative activity in EzBuffers

EzBuffer I: 75%

EzBuffer II: 100%

EzBuffer III: 75%

EzBuffer IV: 100%

FastCut Buffer: 100%

 

Note

It is not sensitive to dam, dcm or CpG methylation. Less than 50% of cleaved fragments are ligated in 20-μl reaction mixtures containing 100-500 units of T4 DNA ligase.

 

● Material Safety Data Sheet

Enzynomics_MSDS_R042_Afl II


● 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.

 

Blue/white screening

To test the integrity of DNA ends, a plasmid, pSKM2 containing a unique site in the lacZ alpha gene is digestesd 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.

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