If you think about the fact that you have the two molecules diffusing randomly through solution, what is the probability that the end of molecule 1 will touch the end of molecule 2? At low concentrations that probability is very low, at high concentrations the probability will go up.

At low DNA concentrations intramolecular ligations are more probably whereas at high DNA then intermolecular ligations are more probable.

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

The efficacy of the ligation step in molecular cloning is significantly influenced by the concentration and volume of the DNA inserts and plasmids. For large inserts and plasmids, optimizing the molar ratio is crucial, typically a 3:1 insert to vector ratio, which enhances the probability of successful ligation by increasing intermolecular interactions. However, excessively high concentrations can lead to increased viscosity and potential formation of undesirable multimers, as indicated in similar studies utilizing various cloning techniques [1][2]. Conversely, low concentrations may result in inefficient ligation. Therefore, careful optimization of both concentration and volume based on DNA fragment sizes is essential for successful cloning.

Reference

[1]

Wick, R., Judd, L., Wyres, K., & Holt, K. (2021). Recovery of small plasmid sequences via Oxford Nanopore sequencing. Microbial Genomics, 7.

[2]

Stevenson, J., Krycer, J., Phan, L., & Brown, A. J. (2013). A Practical Comparison of Ligation-Independent Cloning Techniques. PLOS ONE, 8.

The concentration of insert and vector is a critical step for ligation. The ligation is based on the number of insert molecules coming in vicinity of the digested vector. The more the reaction is diluted, the chances of molecules coming closer will be reduced, decreasing the chances of ligation. I used a 20uL reaction for a 1kb insert and 6kb vector, and they worked fine for me. I think in your reaction may be done in a 10uL reaction if you have enough concentrated nucleic acids, else 20uL should work fine.