DNA ligation

(KA Longo, 1/01)

    1. Quantitate your cut vector DNA and insert using UV spectroscopy. If your vector has been cut with a single enzyme, you'll want to treat it with Calf Intestinal Alkaline Phosphatase (CIP) first.

    2. Set up the following reaction:

* The amount of insert DNA used will depend on the relative size of the insert compared to the size of the vector. A molar ratio between insert and vector should be calculated.

Typically, the insert/vector ratio for sticky-end ligations ranges between 1:1 and 3:1. For blunt-ended ligations, the insert/vector ratio should be at least 10:1.

The number of moles for vector and insert can be calculated as follows:

(g DNA)


[(# of base pairs DNA) x (660 daltons/bp)]

You can use the following equality to figure out the amount of insert DNA needed for a known amount of vector in a blunt-end ligation:

(g vector DNA) (g insert DNA)

----------------------------------------- x 10 = -----------------------------------------

[(# of bp vector DNA) x (660 daltons/bp)] [(# of bp insert DNA) x (660 daltons/bp)]


10 x (g vector DNA) x (# of bp insert DNA)

g insert DNA = ----------------------------------------------------------

(# of bp vector DNA)

Note that any desired ratio can be substituted for the number 10 in the above equation, depending on the type of ligation you are performing.