Question

Your ligation involved combining 100ng od digested plasmid with 30ng of digested insert, yet this corresponds to a molar ratio of approximately 1:5 (plasmid to insert). Explain the difference between ng of DNA and molar ratio.

Answer 1

ng of DNA refers to the total mass of the DNA in solution, while the molarity of the DNA molecule also accounts for the size of the DNA molecule.

For a given DNA molecule, the mass of DNA required to make 1 mol of DNA will differ based on the size of the DNA molecule. For small molecules, because the mass of a single molecule is very less, less mass is required to make 1 mol, of that DNA molecule. However, if a DNA molecule is large, more mass will be reuired to obtain the same number of moles. Conversely, for a large DNA molecule, 1 mole of DNA will have more mass than a small molecule.

Suppose the insert DNA is 1000 bp long and the vector backbone is also 1000 bp long. In such a case a 1:1 ng ratio corresponds to a 1:1 molar ratio. However, if the insert is say 1000 bp and the vector backbone is 2000 bp, a 1:1 ng ratio corresponds to a 1:0.5 or 2:1 molar ratio.