Description
T4 DNA Ligase MBG is an ATP-dependent recombinant enzyme isolated from Escherichia coli strain used to clone DNA. T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA. It will join both blunt-ended and cohesive-ended restriction fragments of DNA and repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids. It is supplied with 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 50% (v/v) glycerol. One (Weiss) unit of T4 DNA Ligase catalyzes the conversion of 1 nmol of 32P from pyrophosphate into Norit-adsorbable material in 30 minutes at 37°C. One Weiss unit is equivalent to approximately 200 cohesive end units.
Assay Specification DNase contamination None detected Exonuclease activity None detected Endonuclease activity None detected
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA. It will join both blunt-ended and cohesive-ended restriction fragments of DNA and repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids. The 10x Ligation Buffer and ATP Solution should be thawed and resuspended at room temperature. For blunt-end ligations, use higher quantities of both the vector and the insert DNA. For sticky cohesive-end ligations, we recommend heating both the vector and the insert DNA before ligation. The electrotransformation efficiency may be improved by heat inactivation of the T4 DNA Ligase MBG and purification of the DNA through a spin column purification method. We recommend using a 3–10 molar excess of insert DNA over vector DNA. The enzyme is inhibited by >200 mM NaCl or KCl concentrations. Inactivate the enzyme at 65°C for 10 minutes or at 70°C for 5 minutes.
Quality Control T4 DNA ligase activity is assayed in a reaction containing 1 µg of bacteriophage lambda DNA digested with HindIII, 1x T4 Ligation Buffer and varying amounts of enzyme for 20 minutes at 16°C. Results are assayed by agarose gel electrophoresis. The product is free of unspecific DNA nucleases. Exonuclease and endonuclease activities were evaluated by gel electrophoresis following incubation of 1 µg of DNA with enzyme in a 50 µL volume for 4 hours at 37°C.
