How did I get to know the DNA Ligation Kit from Takara? Around 2 years ago, I was trying to do a cloning and I was stuck at the ligation step. At first, I was using the regular T4 DNA ligase and attempted to perform ligation several times with this enzyme, but couldn’t get any clones after transformation. I discussed this with the post-doc in the lab. He then suggested that I try the Takara kit. Amazingly, I got positive colonies with the first reaction. I have used this kit for all my cloning since then. The Takara DNA Ligation Kit is a two-component system which includes a T4 DNA ligase and an optimized buffer system. Solution A is the reaction buffer and Solution B is the enzyme. The solutions should be stored in -20ºC; they can be thawed and frozen repeatedly, which saves the trouble of aliquoting. The ligation efficiency is much higher that regular T4 ligase, so generally a 30 min reaction may be enough, although an overnight reaction improves the efficiency.
For the ligation reaction, I used double-digested, dephosphorylated plasmid DNA and double-digested cDNA insert. The protocol recommends using 100 mM Tris-HCl, 7.6 and 5 mM MgCl2 for suspension of DNA. For my experiments, I always use TE buffer, which works fine. The total combined volume of the vector DNA and insert DNA should be around 5-10 µl, and then add 4-8 volumes of solution A and 1 volume of solution B. The reaction temperature is 16 ºC. Higher temperature will decrease the efficiency. For maximum ligation efficiency, researchers may try different insert:vector molar ratios. Generally, a ration of 3:1 has the highest efficiency. In the kit protocol, there is a table comparing the efficiency differences of various molar ratios for both the Takara kit and regular T4 DNA ligase.
What can you do if the ligation efficiency is low? First, try to extend the reaction time to overnight. Secondly, adjust the ratio between the insert and vector. Thirdly, adjust the salt concentration in the ligation mixture. If you try all these and still can’t get good ligation results, then you may want to go back to check if the vector DNA and insert DNA were purified properly and are in good condition.
In addition to DNA insertion into plasmid vector, the Takara DNA Ligation Kit also can be used for ligation of DNA into λ phage vectors, self-circulation of linear DNA, etc. The kit provides detailed protocols for different applications.
In summary, Takara’s DNA Ligation Kit provides a very simple method for quick and efficient ligation reactions.