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Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids. T4 DNA Ligase requires ATP as a cofactor. Highlights • Active in Themo Scientific restriction enzyme, PCR, and RT buffers (when supplemented with ATP) • Fast—sticky-end ligation is completed in 10 minutes at room temperature • Supplied with PEG solution for efficient blunt-end ligation Applications • Cloning of restriction enzyme generated DNA fragments • Cloning of PCR products • Joining of double-stranded oligonucleotide linkers or adaptors to DNA • Site-directed mutagenesis • Amplified fragment length polymorphism (AFLP) • Ligase-mediated RNA detection (see Reference 3) • Nick repair in duplex DNA, RNA or DNA/RNA hybrids • Self-circularization of linear DNA. Includes • T4 DNA Ligase • 10X T4 DNA Ligase Buffer • 50% PEG Solution Notes • Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis. • The volume of the ligation reaction mixture should not exceed 10% of the competent cell volume in the transformation process. • Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction. The extracted DNA can be further precipitated with ethanol |
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