Question

Describe ligation reaction. Discuss the possible products of ligation reaction.What is the maximum transformation efficiency that can be expected? What things can influence the transformation efficiency and ligation reaction using 2 endonuclease for subcloning instead of one?

Answer 1

Ligation

The desired gene of interest irrespective of species, can be expressed (as protein ) in large quantity by harnessing the E. coli bacterium as host with help of recombinant DNA technology.

A process used to join two DNA fragments in molecular cloning process by formation of phosphodiester bonds.

The product of cloning results in the clone ( gene of interest DNA+ vector DNA), which can be transformed into E. coli for expression of our gene of interest.

The transformation efficiency can be defined by number of colony forming unit (cfu) can be given by the formula

Transformation efficiency =colonies found in the plate / concentration of DNA used/ dilution of DNA used for plating.

Factors affecting transformation

1. Bacterial strains used
2. Growth of the bacterium
3. Media composition of the medium used for bacterial growth
4. concentration of the DNA used

The choice of using 2 endonucleases while sub-cloning inorder to excise the desired DNA fragment fully from the vector DNA. The ligation reaction joins the two nicks present in both gene of interest and vector DNA.