Question

Pseudomonas putia is thought to degrade hydrocarbons due to the fact that it harbours a TOL plasmid. To verify this assertion, a student was tasked to isolate and grow this soil-dwelling bacteria and to also isolate the plasmid from the microbe. The TOL plasmid is also intended to be used as a cloning vector.
In an attempt to clone a gene into the TOL plasmid, it was observed that the restriction product of the gene of interest were blunt ended, thereby reducing the ligation efficiency of this gene and the TOL plasmid. Elucidate two methods that could be employed to increase the ligation efficiency of your restriction products.

Answer 1

Use a restiction enzyme that produce blunt end cuts in the TOL plasmid, so the plasmid also will be having blunt ends. Inorder to reduce the self ligation, blunt ends of the TOL plasmid dephosphorylate the linear TOL plasmid with alkaline phosphatase. Phosphorylate the gene of interest with T4 polynucleotide kinase, so that there is 5' Phosphate end to aid ligation with the plasmid.

Or Amplify the gene of interest with Primers in such a way that the amplified gene of interest will have overhangs or sticky ends . Then cut TOL plasmid with the restriction enzyme that makes the same sticky end cuts as the one present in gene of interest so that the gene of interest and TOL plasmid will have same sticky ends that helps in their ligation.