6. You want to ligate a 0.5kb DNA insert into a 2kb vector. You have digested vector at an estimated concentration of 20ng/l and digested insert at an estimated concentration of 30ng/l. The final reaction mixture should contain 40ng (2 l) 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 l of the DNA ligase enzyme and should be made up to a final volume of 20l with MQ water. Complete the recipe below.

2 l vector

_ l insert

_ l 10X ligation buffer

0.2 l DNA ligase

_ l MQ water

20 l total volume

Remember: m = n x Mw Where m = mass (g) n = moles (mol) Mw = molecular weight (g/mol) n = C x V Where n = moles (mol) C = concentration (mol/L, M) V = volume (L) C1xV1 = C2xV2 Where C1 = initial concentration V1 = initial volume C2 = final concentration V2 = final volume (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 dont need to know what this Mw is to answer this question, knowing the length of each piece of DNA is enough!