bro, i have problem with ligation of my gene. Transformation and ligation of control product as well as puc dna given with kit is going well. But when my topo ligation reaction take place it shows no colony on plate. I have incubate at 22 degree celsius from 30 min to overnight but still there is no growth of colony with my insert. I have even checked the primer twice but they are also accurately designed with CACC at ends. What can be the best method for cloning of such large fragment with this kit?