Determining DNA concentrations for ligations

Contributed by Ivan Delgado <>

Calculate DNA concentration for efficient DNA ligations.

  • As an example, for any given ligation, use the following formula:

amount of insert = insert size / vector size x 3 (molar ratio of insert / vector) x amount of vector to be used

In the case of a 4.0 kb sized vector (after proper digestion) and a 0.4 kb sized fragment:

In order to use 1 mg of vector (cohesive end ligation), the following calculations are needed:

insert size / vector size = 0.4 / 4 = 0.1 (1/10) molar ratio of insert / vector (since this is a cohesive end ligation with two different ends, there is no problem with multiple inserts, so a high ratio of insert to vector is possible -for blunt end ligations this ratio should be much lower, even 1:1) = 3/1 Therefore, for a ligation between a 4.0 kb vector and a 0.4 kb fragment, starting with 1 ug of vector, we get:

amount of insert = 0.4/4.0 x 3 (3/1) x 1 ug amount of insert for 1 mg of vector = 0.9 ug

If this had been a blunt end ligation, a 1:1 ratio would have been used and the amount of insert necessary would have been 0.3 ug

These calculations were made for the equivalent of 10 units of ligase

This method is based, with permission, on an original protocol available here.