Qiagen, EN11-500, T4 DNA Ligase MBG (5000 U)

10 x 100 μL of T4 DNA Ligase 5U/ μL, 10 x 200 μL 10x T4 Ligation Buffer, 10 x 80 μL of ATP Solution (25mM), 10 x 200 μL of 50% PEG Solution Storage temperature: Keep at -20°C.

Features

- Catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini
- Exhibits very fast and efficient ligation of DNA fragments with compatible cohesive or blunt ends

Product Details

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.

Performance

Assay: Specification
DNase contamination: None detected
Exonuclease activity: None detected
Endonuclease activity: None detected

Principle

- 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.

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.

Procedure

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.

Applications

- Molecular cloning of PCR products or restriction fragments
- Site-directed mutagenesis
- Nick repair in duplex DNA, RNA or DNA/RNA hybrids
- Self-circularization of linear DNA
- LM PCR methods (Ligation Mediated PCR)

This is used for applications such as: