T4 DNA Ligase is an ATP-dependent recombinant enzyme, isolated from Escherichia coli, used for DNA cloning. 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 as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
- Cloning of PCR products
- Cloning of restriction fragments
- Joining of double-stranded oligonucleotide linkers or adaptors to DNA
- Site-directed mutagenesis
- Nick repair in duplex DNA, RNA or DNA/RNA hybrids
- Self-circularization of linear DNA
- LM PCR methods (Ligation Mediated PCR), eg. Amplified fragment length polymorphism (AFLP)
10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 50 mM KCl, 50% (v/v) glycerol
10x Ligation Buffer
660 mM Tris-HCl (pH 7.5), 100 mM DTT, 100 mM MgCl2
The 10x Ligation Buffer does not contain ATP, which must be added separately. For most applications, ATP should be added to the reaction to a final concentration of 0.5-1.0 mM. The 25 mM ATP Solution is included.
Inhibition and Inactivation
Heat inactivated at 65°C for 10 minutes or 70°C for 5 minutes
One (Weiss) unit of T4 DNA Ligase catalyzes the conversion of 1 nmol of 32P from pyrophosphate into Norit-adsorbable material in 20 minutes at 37°C.
T4 DNA Ligase is functionally tested in cloning assays and is free of detectable contaminating nuclease activities. The following QC assays are performed on each new lot:
- Endonuclease Activity (Nicking) assays
1 µg of supercoiled pUC19 plasmid DNA is incubated with 50 U of T4 DNA Ligase in 1× Ligation Buffer for
4 hours at 37°C. Following incubation, the DNA is visualized on an agarose gel. There must be no visible nicking or cutting of the DNA.
- Non-Specific DNase Activity assay
The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
- Functional assay
Linearized pUC19 plasmid (leaving either blunt-end or cohesive ends) is re-ligated with 5 U of T4 DNA Ligase. The DNA is then transformed into E. coli competent cells that are plated on ampicillin plates. The re-ligation efficiency is determined by counting transformed bacterial colonies.
T4 DNA Ligase is shipped on dry or blue ice.
Storage and Stability
All components should be stored at -20°C. When stored under optimum conditions, the reagents are stable for a minimum of 12 months from date of purchase.
1. Weiss, B. et al., Enzymatic breakage and joining of deoxyribonucleic acid. J. Biol. Chem. 243, 4543-4555 (1968).
2. Pheiffer, B.H. et al., Polymer-stimulated ligation: enchanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853-7871 (1983).
3. Rossi, R. et al., Functional characterization of the T4 DNA Ligase: a new insight into the mechanism of action. Nucleic Acids Res. 25, 2106-2113 (1997).
4. Nilsson, M. et al., RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res. 29, 578-581 (2001).
5. Cherepanov, A.V. et al., Binding of nucleotides by T4 DNA Ligase and T4 RNA Ligase: optical absorbance and fluorescence studies. Biophys. J. 81, 3545-3559 (2001).
Manufacturer: BLIRT S.A.