Description

• Application: Suitable for:Ligation of blunt ended or cohesive DNA fragmentsLigation of cloning vector and restriction insert fragmentsSeal nicks in double stranded DNA and RNA or DNA/RNA hybridsCouple RNA single strands by bridging oligonucleotide adapters
• Biochem/physiol Actions: T4 DNA Ligase forms an energy dependent phosphodiester linkage between the termini of adjacent polynucleotides of duplex DNA. The ligation reaction requires ATP as a cofactor.[1] Ligation of blunt-ended fragments requires higher enzyme concentration and can be facilitated by using PEG in the reaction mixture.[2] The enzyme requires a 3′ hydroxyl and 5′ phosphate for ligation. Self-ligation of vector DNA can be prevented by dephosphorylation with alkaline phosphatase. T4 ligase plays an active role in repair of DNA and RNA nicks.[3]
• Components: T4 DNA Ligase is supplied in a solution containing 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, and 50% (v/v) glycerol.
• Unit Definition: One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmole of P32 from pyrophosphate into ATP as Norit-absorbable material in 20 minutes at 37°C.
• Other Notes: T4 DNA Ligase is inactivated by heating at 65 °C for 10 minutes.