Name: T4 DNA Ligase
Brand: ArcticZymes Technologies

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. It is also completely inactivated by incubating at 70°C for 10 minutes.

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.

Key Features

ATP and Mg2+ dependent dsDNA ligase
Easily heat-inactivated at 70°C for 10 minutes
Extensively tested for contaminating DNase and RNase activities as well
as residual host-cell gDNA

Applications

Ligation of dsDNA
NGS library prep
Molecular cloning

Figures

Figure 1. No residuals of E.coli gDNA were detected in ArcticZymesT4 DNA Ligase

T4 DNA Ligase activity on various substrates. In vitro assays of ligation activity were performed using three oligos hybridised into a nicked 20 bp dsDNA substrate. Efficient DNA ligation was observed after 30 min at 25°C when using 1.12 U/μl T4 DNA Ligase. At 0.373 U/μl T4 DNA Ligase showed about 70% substrate turnover.

Figure 2. T4 DNA Ligase displays good nick-joining activity of dsDNA

T4 DNA Ligase activity on various substrates. In vitro assays of ligation activity were performed using three oligos hybridised into a nicked 20 bp dsDNA substrate. Efficient DNA ligation was observed after 30 min at 25°C when using 1.12 U/μl T4 DNA Ligase. At 0.373 U/μl T4 DNA Ligase showed about 70% substrate turnover.

Properties

Unit Definition

0.1 units is defined as the amount of enzyme that is needed to convert 1 pmol (of 18 pmol) of nicked DNA substrate in 20 minutes at 25 °C in a 20 µl reaction volume in a buffer consisting of 62.5 mM Tris-HCl pH 7.5, 10 mM DTT, 1 mM ATP, 0.05 mg/ml BSA and 25 mM KCl.

One Weiss Unit is equivalent to approximately 500 ArcticZymes Units.

Size

56.9 kDa

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 2 years in the supplied storage buffer.

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

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.a

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 (1 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).