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T4 DNA Ligase MBG (500 U)
Cat. No. / ID: EN11-050
100 μL of T4 DNA Ligase 5U/ μL, 200 μL 10x T4 Ligation Buffer, 80 μL of ATP Solution (25mM), 200 μL of 50% PEG Solution.
Storage temperature: Keep at -20°C.
Quantity
500 U
2500 U
5000 U
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The T4 DNA Ligase MBG is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
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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
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.
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
This is used for applications such as:
- 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)
Resources
Certificates of Analysis (1)