T4 DNA Ligase

Overview

T4 DNA Ligase is an ATP and Mg2+ dependent dsDNA ligase which catalyses the formation of a phosphodiester bond between 3’-hydroxyl and 5’-phosphate termini in duplex DNA, duplex RNA and some DNA/RNA hybrids. T4 DNA Ligase is active on both blunt-end and cohesive-end substrates.

This is a high-quality (commercial grade) version of the T4 DNA Ligase. T4 DNA Ligase is recombinantly produced in E. coli. ArcticZymes’ T4 DNA Ligase is extensively tested for contaminating DNase and RNase activities as well as residual host-cell gDNA.

Contaminating E. coli gDNA in T4 DNA Ligases
Residual E. coli gDNA in commercial T4 DNA Ligases
Figure 1: T4 DNA Ligases often contain E. coli gDNA. ArcticZymes’ T4 DNA Ligase and four commercially available T4 DNA Ligases (5000 Units) were assayed for contaminating E. coli gDNA. E. coli gDNA was detected in two of the commercial ligases. Assays were run in triplicates using a probe-based qPCR assay.

Properties

Unit Definition: One unit is defined as the amount of enzyme required to give 75% ligation of the 2322 and 2927 bp HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300 µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP and 10 mM DTT.

One Weiss Unit is equivalent to approximately 500 ArcticZymes Units.

Inactivation: T4 DNA Ligase can be completely inactivated by incubating at 70°C for 10 minutes.

Storage and Stability

The enzyme is stable at -20°C for > 1 year in the supplied storage buffer.

The enzyme tolerates at least four freeze-thaw cycles (-80°C) without loss of activity.

Quality Control

ArcticZymes is dedicated to the quality of our products. T4 DNA Ligase is manufactured at our ISO 13485 certified facility in Norway.

dsDNA endonuclease activity

10 000 U T4 DNA Ligase was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.

ssDNA endonuclease activity

10 000 U T4 DNA Ligase was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.

Exonuclease activity

10 000 U T4 DNA Ligase was incubated with either 3H-dATP labelled ds or ssDNA (0.5 µg, 500 bp) for 4 hours at 37°C. Acid soluble radioactivity from labelled DNA was not significantly over blank test for either substrate.

RNase activity

5000 U T4 DNA Ligase was incubated with a 2 kb RNA transcript (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of RNA degradation.

E. coli gDNA contamination

5000 U T4 DNA Ligase was analysed in a probe-based qPCR assay detecting the 23S ribosomal subunit in E. coli. No E. coli gDNA could be detected (LOD: < 3 E. coli genomic copies).

Additional Data and Information

We are pleased to provide additional data relating to T4 DNA Ligase upon request.

We appreciate suggestions for product modifications.

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