T4 DNA Ligase

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5’ phosphate and 3’ hydroxyl termini in duplex DNA or RNA.

Recommended Reaction Condition: Overnight ligation at 16°C with 0.3-1 µl of the enzyme per 10-20 µl reaction. It is known that phenol-chloroform extraction, followed by ethanol precypitation of ligated DNA, improves yield of transformation.

Source: T4 bacteriophage of Escherichia coli

Description:

· Catalyzes the joining of duplex DNA molecules at blunt ends (1).

· Covalently joins DNA fragments with complementary cohesive ends.

· Seals single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

· Ultrapure recombinant enzyme.

· Suitable for cloning of restriction fragments and joining linkers or adapters to blunt-ended DNA (2).

Unit Definition: One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5' DNA termini concentration of 0.12 µM [300 µg/ml]) in 20 μl of 1 x T4 DNA Ligase Reaction Buffer in 30 minutes at 16°C.

Note: 67 cohesive end ligation units are the equivalent of one Weiss unit.

Storage Conditions: Store at -20°C.

1 x Reaction buffer: 50 mM Tris-HCl (pH 7.5 at 25°C), 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP, 25 µg/ml BSA.

Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 22°C), 1 mM dithiothreitol, 50 mM KCl and 50% (v/v) glycerol.

Assay Conditions: 66 mM Tris (pH 7.6 at 22°C), 6.7 mM MgCl₂, 67 mM ATP, 10 mM dithiothreitol, 3.3 µM [a-³²P]Na₄P₂O₇. Reaction volume is 300 µl.

Quality Control: All preparations are tested for contaminating endonuclease, exonuclease and non-specific single- and double-stranded DNase activities. The preparation is approximately 95% pure as judged by SDS polyacrylamide gel electrophoresis.

References:

1. Weiss, B., Jacquemin-Sablan, A., Live, T.R., Fareed, G.C. and Richardson, C.C. (1968) J. Biol. Chem. 243, 4543-4555.

2. Sambrook, J. et al. (1989) Molecular cloning: A laboratory Manual, second edition, pp. 1.53-1.73, Cold Spring Harbor, New York.