SKU: 3250742408

T4 DNA Ligase

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Description

T4 DNA LigaseProduct Specification Synonyms DNA LigasePolydeoxyribonucleotide synthase [ATP] Amino Acid Sequence Expression System E. coli Molecular Weight 57 kD Purity 95% by SDS PAGE & RP HPLC Conjugation Unconjugated Tag His Tag Physical Appearance Liquid Storage Buffer 10 mM Tris HCl50 mM KCl1 mM DTT0. 1 mM EDTA50% Glycerol pH 7. 4 @ 25C Stability & Storage Store at 25 ~ 15 for 1 years Reference [1] Williamson A, Pedersen H. Recombinant expression and

Product Specification


Synonyms DNA Ligase、Polydeoxyribonucleotide synthase [ATP]
Amino Acid Sequence


Expression System E.coli
Molecular Weight

57 kD

Purity >95% by SDS-PAGE & RP-HPLC
Conjugation Unconjugated
Tag His Tag
Physical Appearance Liquid
Storage Buffer

10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol (pH 7.4 @ 25°C)

Stability & Storage

Store at -25 ~ -15℃ for 1 years

Reference

[1] Williamson A, Pedersen H. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida. Protein Expr Purif. 2014 May; 97:29-36.
[2] Liu X, Huang A, Luo D, Liu H, Han H, Xu Y, Liang P. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli. Protein Expr Purif. 2015 May;109:79-84. Epub 2015 Feb 17.

Background

T4 DNA ligase is a type of DNA ligase. DNA ligase catalyzes the formation of phosphodiester bonds at single-stranded DNA breaks in double-stranded DNA in vivo. DNA ligase has important biological functions in organisms. In DNA repair and recombination, DNA ligase plays a role in connecting gaps. In the process of DNA replication, the synthesis of the lagging strand is discontinuous, and DNA ligase connects the discontinuous DNA strand into a continuous DNA strand. It does not contain DNA endonuclease, exonuclease and phosphatase, and does not contain RNA enzyme.

Components

Storage Solution: 400U/μl T4 DNA Ligase、10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol (pH 7.4 @ 25°C) 10*Reaction Buffer: 500 mM Tris-HCl、100 mM MgCl2、10 mM ATP、100 mM DTT (pH 7.5 @ 25°C)

Protocol

1、Set up the following reaction in a microcentrifuge tube on ice. Add the following components in sequence. Note that the table shows a ligation using1. a molar ratio of 1:5 vector to insert for the indicated DNA sizes.

Components

Volume 20μl

10* T4 DNA Ligase Buffer

2 μl

Vector DNA (such as pET-28a 5369bp)

50ng

Insert DNA (530bp)

25ng

Nuclease-free water

Up to 20 μL

T4 DNA Ligase

1μl

2. Gently mix the reaction through the up and down pipette, and inhale the liquid briefly.

3. For sticky ends, incubate at 16°C overnight or at room temperature for 10 minutes.

4. For blunt ends or single base overhangs, incubate overnight at 16°C or room temperature for 2 hours (alternatively, high concentrations of T4DNA ligase can be used for 10 minutes of ligations).

5. Chill on ice and transform 1-5 μl of the reaction into competent cells.

Guidelines

1. ATP is an essential cofactor in the reaction. This is in contrast to E. coli DNA Ligase, which requires NAD as a cofactor. 

2. If T4 DNA Ligase is to be diluted, it is recommended that it be diluted with 50% glycerol in storage buffer and stored at -20°C. 3. Room temperature ligation

Unit Definition

One unit is the amount of enzyme required to ligate 50% of the HindIII-digested λ DNA fragments [DNA 5´-end concentration of 0.12 µM (300 μg/ml)] within 30 minutes at 16°C in a 20 µl reaction system and in 1X T4 DNA Ligase Reaction Buffer.
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SKU: 3250742408

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