T7 DNA Ligase

• Join blunt-end and cohesive-end termini
• Repair single-stranded nicks in duplex DNA

T7 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5ʹ phosphate and a 3ʹ hydroxyl termini in duplex DNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA.

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

2X Rapid Ligation Buffer (cat. no. B1010) contains the following: 132 mM Tris-HCI, 20 mM MgCl₂, 2 mM DTT, 2 mM ATP and 15% PEG 6000; pH 7.6 at 25°C.

SDS available on request.

Enzyme properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein

The protein is produced by a recombinant E. coli strain carrying the T7 DNA Ligase gene.

Unit definition: One unit is defined as the amount of T7 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.

Molecular weight: 41,132 Daltons

In the absence of high concentration crowding agents (e.g., 20–30% PEG 6-8000), the specific activity of T7 DNA Ligase is reduced by 1000-fold on blunt-ended fragments (1, 2).

T7 DNA Ligase is incapable of ligating single-stranded DNA fragments (1).

References

1. Doherty, A. et al. (1996) J.Biol. Chem 271:11083.
2. Dayal, T. Unpublished observations.

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X Rapid Ligation Buffer and added to 20 µl reactions containing double-stranded DNA fragments and 1X Rapid Ligation Buffer. Reactions were incubated 30 minutes at approximately 23°C (room temp), placed on ice, and analyzed on a 1% agarose gel stained with ethidium bromide.

Protein concentration is determined by OD₂₈₀ 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 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.

• Cloning igation
• Adding linkers or adapters to double-stranded DNA
• Nick-sealing in double-stranded DNA