Pfu DNA Ligase

Description

Description:

Pfu DNA Ligase is a heat-stable enzyme that facilitates the creation of a phosphodiester bond between the 5´-phosphate and the 3´-hydroxyl groups of two neighboring DNA strands that are fully paired and aligned with a complementary DNA strand, ensuring no gaps. This ligase requires ATP as a cofactor and exhibits activity within a range of elevated temperatures, from 45°C to 70°C.

Pfu DNA Ligase is highly effective for ligating nicks in DNA during incubations at high temperatures. Its extreme thermostability allows it to withstand the rigorous conditions of PCR processes. While it does not ligate short 4 base overlaps, which are common in restriction enzyme digests, it is proficient at efficiently ligating 12 base pair overlaps. Additionally, this enzyme is isolated from a recombinant source, ensuring its purity and effectiveness for precise molecular biology applications

Applications:

Pfu DNA Ligase will ligate this substrate:

• Detect allele-specific genes using Ligase Detection Reaction and Ligase Chain Reaction^(1,2)
• Incorporate a phosphorylated oligonucleotide during PCR amplification for mutagenesis⁽³⁾

Source:

Purified from an E. coli strain that harbors the cloned ligase gene.

Reagents Supplied:

Unit Definition:
(Cohesive End Unit) One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 µl in 15 minutes at 45°C. A cohesive end unit is equivalent to the nick-closing unit(1).

Supplied with:
10X Pfu DNA Ligase Reaction Buffer

10X Pfu DNA Ligase Reaction Buffer
10 mM Tris-HCl
600 µM ATP
2.5 mM DTT
2.5 mM MgCl₂
0.1% Triton® X-100
(pH 7.5 @ 25°C)

Supplied in:
Storage Buffer:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml Recombinant Albumin
50% Glycerol
10 mM ammonium sulfate
pH 7.4 @ 25°C

Reaction Conditions:
1X Pfu DNA Ligase Reaction Buffer
Incubate at 45°C

Usage Concentration: 40,000 units/ml

Heat Inactivation: No

Unit Assay Conditions:
In a 50 µl reaction mixture containing 1X Pfu DNA Ligase Reaction Buffer and 20 µg/ml BstEII-digested λ DNA, the components were incubated at 45°C for 15 minutes. To stop the reaction, stop dye (composed of 50% glycerol, 50 mM EDTA, and bromophenol blue) was added, followed by heating the mixture at 70°C for 10 minutes. The treated sample was then loaded onto a 0.7% agarose gel. The presence of ligase in the reaction enables the cos ends of the BstEII-digested λ DNA to remain joined together even after the 70°C heat treatment.

Recommended Storage Condition: -20°C

References:

1. Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88, 189-193.
2. Barany, F. (1991) The Ligase Chain Reaction in a PCR World. (pp. 5-16, Cold Spring Harbor: Cold Spring Harbor Laboratory).
3. Michael, S.F. (1994) Biotechniques 16, 411-412.

Additional information

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