Description

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. 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. +BP12Storage 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 (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).