Qiagen, L6060L, Taq DNA Ligase (20,000 U)

Taq DNA Ligase (40,000 U/mL) and 10X Taq DNA Ligase Buffer

Features

- Thermostable DNA Ligase
- Seals nicks
- Discriminates against mismatch ligation

Product Details

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 (1).

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 at 25°C)

Supplied with: 10X Taq DNA Ligase Buffer (B6060): 200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl 2 , 5 mM NAD + , 0.1% Triton X-100 (pH 7.6 at 25°C)

Performance

Test: Amount tested
Purity: n/a
Specific activity: n/a
Single-stranded exonuclease: 400 U
Double-stranded exonuclease: 400 U
Double-stranded endonuclease: 400 U
E. coli DNA contamination: 400 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 76.9 kDa

Principle

The enzyme is produced by a recombinant E. coli strain carrying the cloned Taq DNA Ligase gene.

One 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 SmaI and SalI in 50 µL 1X Taq DNA Ligase Buffer following a 10-minute incubation at 45°C.

Procedure

Components: Final Concentration
Nuclease-free water: N/A
10X Taq DNA Ligase Buffer (B6060): 1X
DNA: up to 1µg
Taq DNA Ligase (L6060L): 80 U
Total Volume =: 50 µL

- Set up the following reaction mixture in a total volume of 50 µL: Components Final Concentration Volume Nuclease-free water N/A X µL 10X Taq DNA Ligase Buffer (B6060) 1X 5 µL DNA up to 1µg X µL Taq DNA Ligase (L6060L) 80 U 2 µL Total Volume = 50 µL
- Incubate at 45°C for 10 minutes.

Usage Instructions

Nick ligation in double-stranded DNA

Quality Control

Unit activity is measured using a 2-fold serial dilution method. Dilutions of the 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.

Protein concentration (OD 280 ) is determined by OD 280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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.

Applications

- High-temperature ligation
- Ligase chain reaction
- Ligase detection reaction

- Takahashi, M. et al. (1984). J. Biol. Chem. 259, 10041-10047.

This product is available for molecular biology applications such as:

References