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Taq DNA Ligase

Product Description

Taq DNA Ligase catalyzes the formation of a phosphodiester bond in duplex DNA containing adjacent 5′-phosphoryl and 3′-hydroxyl termini, using NAD+ as a cofactor.

Source of Protein
A recombinant E. coli strain carrying the cloned Taq DNA Ligase gene.

Supplied in
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
0.1% Tween-20
50% glycerol
pH 7.5 @ 25°C

Supplied With
B6060 (10X Taq DNA Ligase Buffer)

10X Taq DNA Ligase Buffer (B6060)
200 mM Tris-HCI
250 mM KCl
100 mM MgCl2
5 mM NAD
0.1% Triton X-100
pH 7.6 @ 25°C

Unit Definition
1 unit is defined as the amount of Taq DNA Ligase required to join 50% of 1 µg of the 12-base cohesive ends of Lambda DNA cut with Sma I and Sal I in 50 µl 1X Taq DNA Ligase Buffer following a 10 minute incubation at 45°C.

Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme batch were made in 1X Taq DNA Ligase Reaction Buffer and added to 50 µL reactions containing λ Hind III digested DNA and 1X Taq DNA Ligase Reaction Buffer. Reactions are incubated for 10 minutes at 45°C, stopped, and analyzed on a 0.8% agarose gel stained with ethidium bromide.

SDS-Page (Physical Purity Assessment)
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the same enzyme species. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Contamination Tests

Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
A 50 µl reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.

Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pENZuC DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination test
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values and standard curve data, the detection limit of this assay is <10 copies genome/sample.

Product Information

Taq DNA Ligase
Part NumberL6060L
Price$363
Concentration40,000 U/ml
Unit Size20,000 U
SDS Available on request

Product Specification*

Storage Temperature-25 to -15°C
Test Specification
Purity (SDS-PAGE)>99%
Specific Activity400,000 U/mg
SS Exonuclease400 U <5.0% released
DS Exonuclease400 U <1.0% released
DS Endonuclease400 U = No conversion
E.coli DNA Contamination400 U <10 copies

* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

Legal Disclaimer

Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application; therefore it is the sole responsibility of users of this product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.

Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. MSDS sheets relevant to this product are available upon request.

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