Thermostable Taq DNA Ligase catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphoryl and 3'-hydroxyl cohesive termini in duplex DNA fragments.
Source: Thermus aquaticus
Description:
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Catalyzes the formation of a phosphodiester bond between duplex DNA fragments with cohesive ends.
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Condensation of the 5'-phosphoryl group with an adjacent 3'-hydroxyl group is coupled with the hydrolysis of NAD+.
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Stable at elevated temperatures (45°C-65°C) allowing enhanced hybridization stringency (2).
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Enzyme suitable for:
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allel-specific gene detection using Ligase Detection Reaction and Ligase Chain Reaction (3,4)
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mutagenesis by incoporation of a phosphorylated oligonucleotide during PCR amplification (5).
Unit Definition: One unit catalyzes the ligation of 50% of the cos sites in 0.4 µg of Sma I-and Sal I-digested bacteriophage lambda DNA in 1 minute at 45°C in a 20 µl reaction.
Storage Conditions: Store at –20°C.
Storage Buffer: 20 mM Tris-HCl (pH 7.6 at 22°C), 0.1% (v/v) Brij-35, 50 mM KCl, 0.1% (v/v) Brij-35 and 50% (v/v) glycerol.
Assay Conditions: The activity assay is carried out with 0.4 µg of Sma I- and Sal I digested bacteriophage lambda DNA in a 20 µl volume. The reaction buffer consists of 20 mM Tris-HCl (pH 7.6 at 25°C), 25 mM potassium acetate, 10 mM dithiothreitol, 10 mM magnesium acetate, 0.6 mM NAD+ and 0.1% (v/v) Brij-35. The reaction is followed by agarose gel electrophoresis.
Quality Control: All preparations are assayed for contaminating endonuclease, exonuclease, nonspecific single- and double-stranded DNase activities.
References:
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Barany, F. (1991) PCR Methods and Applications 1, 5-16.
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Wu, D.Y. and Wallace, R.B. (1989) Genomics 4, 560-569.
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Barany, F. (1191) Proc. Natl. Acad. Sci USA 88,189.
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Barany, F. (1991) The Ligase Chain Reaction in a PCR World, Cold Spring Harbor Laboratory Press ISSN pp. 5-16.
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Mischael, S. F. (1994) Biotechniques 16, 411-412.