Lab Protocols‎ > ‎Protocols/Methods‎ > ‎

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:
      Volume (µL)amt. DNA 
    Vector DNA 125-100 ng 
    Insert DNA1* 
    10x T4 DNA ligase buffer 2 
    sterile ddH2O 15 
    T4 DNA ligase 1 
    TOTAL VOLUME 20 

* 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.