6. You want to ligate a 0.5kb DNA insert into a 2kb vector. You have digested ve
ID: 213509 • Letter: 6
Question
6. You want to ligate a 0.5kb DNA insert into a 2kb vector. You have digested vector at an estimated concentration of 20ng/ul and digested insert at an estimated concentration of 30ng/ul. The final reaction mixture should contain 40ng (2 ul) of vector. Ligation is generally most efficient with a 3:1 molar ratio of insert to vector, so you should add enough insert to have an approximate final molar concentration of insert which 3 times higher than the final molar concentration of vector. The ligation mixture should also contain DNA ligase buffer, which is supplied as a 10X stock solution (a 10X stock means the stock solution should be diluted 10 fold in the final mixture), 0.2 ul of the DNA ligase enzyme and should be made up to a final volume of 20ul with MQ water. Complete the recipe below 2111 vector Hint: The Mw of a piece of DNA is approximately proportional to its length because each base pair in a piece of DNA will have approximately the same Mw per bp (660). You don't need to know what this Mw is to answer this question, ul insert ul 10X ligation buffer 0.2 ul DNA ligase ul MQ water 2knowing the lengeth of each piece of DNA is enought 20 ul total volumeExplanation / Answer
The construction of a recombinant plasmid connects the insert DNA (gene or fragment of interest) into a compatibly digested vector backbone. Which is done by covalent connection between the sugar backbones of the two DNA fragments. This reaction is called ligation, is performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which joins the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.
Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration. However, for most standard cloning (where the insert is smaller than the vector) a 3 insert: 1 vector ratio will work just fine. We recommend around 100ng of total DNA in a standard ligation reaction.
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