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.
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.
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.
中文
