Description
Application 0.01 Weiss unit is the amount of enzyme required to ligate >95% of 1 ug bacteriophage lambda Hind III fragments at 16°C in 20 minutes.
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
Features and Benefits • Ligation of blunt ended or cohesive DNA fragments.
• Ligation of cloning vector and restriction insert fragments.
• Seal nicks in double stranded DNA and RNA or DNA/RNA hybrids.
• Couple RNA single strands by bridging oligonucleotide adapters.
Other Notes T4 DNA Ligase is inactivated by heating at 65 °C for 10 minutes.
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.
Physical form Solution in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 50% glycerol （v/v）
Properties
form buffered aqueous glycerol solution
concentration 500-6,000 Weiss units/mL
shipped in dry ice
storage temp. −20°C
Safety
Personal Protective Equipment Eyeshields, Gloves, half-mask respirator （EU）, half-mask respirator （US）, multi-purpose combination respirator cartridge （US）
WGK Germany 3
References
Cited Reference 1. Rusche, J., Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase. Nucleic Acids Res. 13, 1997-2008, （1985） Abstract
2. Upcroft, P., Rapid and efficient method for cloning of blunt-ended DNA fragments. Gene 51, 69-75, （1987） Abstract
3. Engler, M.J. and Richardson, C.C., Boyer, P.D. The Enzymes San Diego, CA , （1982） 5, 3
reference , Sambrook, J., et al. Molecular Cloning: A Laboratory Manual 2nd ed., Plainview, NY , （1989）, 1.53-1.73
Moore, M.J., Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science 256, 992-997, （1992） Abstract
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