Taq DNA Ligase

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

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

This enzyme is supplied in 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Tween-20 and 50% glycerol; pH 7.5 at 25°C.

10X Taq DNA Ligase Buffer (cat. no. B6060) contains the following: 200 mM Tris-HCI, 250 mM KCl, 100 mM MgCl₂, 5 mM NAD and 0.1% Triton X-100; and pH 7.6 at 25°C.

SDS available on request.

Enzyme properties

• Storage temperature: –25°C to –15°C

OEM by QIAGEN offers bulk manufacturing of Taq DNA Ligase in custom formulations

Source of recombinant enzyme protein

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

Unit definition: 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.

Molecular weight: 76,910 Daltons

Unit activity was measured using a twofold 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.

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 band corresponding to the protein of interest 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 activity 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 activity 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 contamination is evaluated using 5 µl replicate samples of enzyme solution that are 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.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

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