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: Even though much less units of the ligase are required for ligation to take place, due to the ligation kinetics for the best cloning results we recommend using 0.3-1 μl of the enzyme per 10-20 μl reaction at 16°C for overnight.
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:
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Catalyzes the joining of duplex DNA molecules at blunt ends (1).
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Covalently joins DNA fragments with complementary cohesive ends.
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Seals single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
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Ultrapure recombinant enzyme.
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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.
Avoid multiple cycles of freezing/thawing of the stock reaction buffer. Thawing should be performed at temperatures not exceeding 10°C. Recommended procedure is to divide the provided reaction buffer into smaller portions.
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 [α-³²P]Na4P2O7. Reaction volume is 30 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:
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Weiss, B., Jacquemin-Sablan, A., Live, T.R., Fareed, G.C. and Richardson, C.C. (1968) J. Biol. Chem. 243, 4543-4555.
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Sambrook, J. et al. (1989) Molecular cloning: A laboratory Manual, second edition, pp. 1.53-1.73, Cold Spring Harbor, New York.