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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.
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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.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM
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
Safety Data Sheets (1)
Certificates of Analysis (1)
Quick-Start Protocols (1)