Question

Explain two methods that can be used to ligate restriction products of a gene of interest with the products having blunt ends

Answer 1

In the 1st method, vector is dephosphorylated to prevent self ligation by the enzyme Phosphatase and the insert (​​​​Desired restriction product with blind ends) needs to be phosphorylated by the enzyme polynucleotide kinase, as the ligation requires the presence of 5'phosphate.

In the 2nd method, the vector need not to be dephosphorylated. In this method, a reaction mixture is prepared with circularized plasmid ( vector ) and insert along with T4 ligase and the restriction enzyme producing blunt end. In this single tube, circular plasmid is cut and insert is ligated. But the empty plasmid ligation also occur along with successful ligations, these T4 empty ligations can be reCut by restriction enzyme which does not act on the plasmid having insert DNA which do not have the restriction site for that enzyme. Therefore empty background signal is lowered by competing Digestion and ligation reactions. And, finally, all the circular plasmids must have the insert.