DNA ligation
(KA Longo, 1/01)
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.
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)]
Or:
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.