1:1 molar ratio of insert to vector is the best for performing Topo clone? Because I just put 1:1 molar ratio (insert to vector) and after 16 hr incubation with LB (kanamycin), I noted one colony in plate. Is this enough to purify the plasmid?
Can we improve the concentration of gene insert rather than another (2:1, 3:1) like this ?
In Topo cloning, a 1:1 molar ratio of insert to vector is commonly used. You You can try increasing the concentration of the insert (2:1 or 3:1 ratio) can certainly help improve cloning efficiency, but it’s also important to consider other factors like transformation efficiency and ligation optimization. You should still be able to purify the plasmid from the single colony, but always verify by sequencing.
You only need 1 colony as long as it's what you want. Don't fuss about with optimizing, just continue with your project.
If the one colony is what you want, then that's good enough for this cloning task. But it's certainly not an ideal situation.
For future cloning, go ahead and try different ratios, or a longer topo reaction time. I'd test the competent cells too, just in case. Oh, and make sure there wasn't anything inhibitory (like loading dye/buffer not included in a topo compatible PCR mix) in your insert. I made that mistake once, and I got 0 colonies.
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