Thermo Scientific™ T4 DNA Ligase (5 U/μL)

Description

• Catalyze the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA with the Thermo Scientific™ T4 DNA Ligase
• 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
• The 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
• 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 by spin column or chloroform extraction
• The extracted DNA can be further precipitated with ethanol.