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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. |
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
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