Ligations require planning. Restriction fragments with protruding ends to be ligated must contain compatible complementary sequences. If orientation of an insert is important, two different ends will increase the probability of the correct orientation. The joining of a blunt end to a sticky end can be achieved by converting the sticky end to a blunt end, either by filling in the missing bases of a 5' protruding end using the Klenow fragment or by chewing off a 3' overhang with T4 DNA polymerase (the polymerase has a 3'-5' exonuclease activity used to correct misincorporation of nucleotides).
The components to be ligated are mixed in a ratio determined by the desired product. If recircularization (intramolecular ligation) is the goal, the concentration of fragments is kept low to decrease the probability of two different molecules contacting each other. If a product is to be inserted, such as in a cloning procedure, an excess of insert of 2 to 3 x the vector concentration is used, and the concentration of DNA is higher to increase the occurrences of intermolecular ligation.
and Intended Use
Created by B. Beason (firstname.lastname@example.org), Rice University, 18 June 2010
Updated 18 June 2010