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Description
T4 DNA Ligase ⅡProduct Specification Synonyms DNA LigasePolydeoxyribonucleotide synthase [ATP] Amino Acid Sequence Expression System E. coli Molecular Weight 81 kD Purity 95% by SDS PAGE & SEC HPLC Conjugation Unconjugated 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 purification of an
Product Specification
| Synonyms | DNA Ligase、Polydeoxyribonucleotide synthase [ATP] |
| Amino Acid Sequence | |
| Expression System | E.coli |
| Molecular Weight | 81 kD |
| Purity | ＞95% by SDS-PAGE & SEC-HPLC |
| Conjugation | Unconjugated |
| 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. |
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. T4 DNA Ligase Ⅱ is a recombinant protein fusion of T4 DNA Ligase and adenylate kinase, The purified T4 DNA Ligase Ⅱ not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. 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 using 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 Ⅱ |
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
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