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分子别名
DNA Ligase、Polydeoxyribonucleotide synthase [ATP]表达宿主
E.coli分子量
57 kD
纯度
＞95% by SDS-PAGE & RP-HPLC活性
400U/μl标记
Unconjugated标签
His Tag性状
Liquid缓冲体系
10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol （pH 7.4 @ 25°C）
储存条件
Store at -25 ~ -15℃ for 1 years
文献引用
[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.
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.
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)
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.
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
- 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.
生物活性
-
The two DNA strands after enzymatic digestion and purification were used as substrate for enzyme linkage and incubated at 25℃ for 2 hours. The reaction was analyzed by 1% agarose gel electrophoresis.
M: DNA marker
Lane 1 negative control
Lane 2 and 3 Experimental result
电泳
2μg (R: reducing condition, N: non-reducing condition).
反相高效液相色谱(RP-HPLC)
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