Dear Muley,

I feel the ratio of vector: insert would be decided depending upon molecular crowding in your reaction mixture. The vector usually is a big molecule and it has two ends cut with restriction enzymes. So, it has a higher chance to reanneal back by circularization. Whereas, the inserts are generally small,so, the chances of inserting the insert in between the two ends of the vector would depend on the number of insert molecules in the ligation mix. More the number of insert, higher is the chance of collision with vector. Hence, higher chance of proper ligation. Thus vector to  insert ratio is ideally 1:3. Depending on the requirement, it can be changed to 1:5 or even 1:7 to increase chances of getting positive clones.

Here is a tool from NEB to calculate the amount of vector and insert depending on molar ratio of your choice.

Hope it helps!

http://nebiocalculator.neb.com/#!/ligation

Dear Muley,

I feel the ratio of vector: insert would be decided depending upon molecular crowding in your reaction mixture. The vector usually is a big molecule and it has two ends cut with restriction enzymes. So, it has a higher chance to reanneal back by circularization. Whereas, the inserts are generally small,so, the chances of inserting the insert in between the two ends of the vector would depend on the number of insert molecules in the ligation mix. More the number of insert, higher is the chance of collision with vector. Hence, higher chance of proper ligation. Thus vector to  insert ratio is ideally 1:3. Depending on the requirement, it can be changed to 1:5 or even 1:7 to increase chances of getting positive clones.

Here is a tool from NEB to calculate the amount of vector and insert depending on molar ratio of your choice.

Hope it helps!

http://nebiocalculator.neb.com/#!/ligation

Dear Tias,

Thank you very much for your response. It helps for sure!

There is an old paper from the eighties or early nineties in NAR or Gene where an algorithm for calculating the optimal vector:insert ratio according to concentration and size is derived analytically. I don't have it here with me and couldn't find it in a quick PubMed search, but I have a hard copy back home. If you want the reference send me a message and I'll get it for you.

That said, in my opinion getting too obsessive about vector:insert ratios is pointless. DNA conc. measurements are not that precise, and you never know the percent of damaged ends in your DNA molecules, so experimentally the best solution always is to try a range of ratios.

Additionally, if there is no nuclease contamination, the ends of the vector and insert are not damaged, the DNA fragments were not heavily irradiated with UV during purification, the ligase is OK and the ligase buffer is OK, it really doesn't matter whether you use one or another molar ratio -you only have to screen a dozen colonies to get recombinant clones.

Thank you so much Alejandro for your response. I agree with the points that you have discussed. I have also observed such scenario and I do use different ratios and then screen most of the colonies.

For anybody interested in the paper I referred to above, it Is PMID 2848221 ("Kinetic analysis for optimization of DNA ligation reactions"). The abstract:

Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for four types of reactions: (a) a homogeneous population of singly restricted DNA fragments, (b) insertion of singly restricted insert into vector, (c) forced directional insertion of doubly restricted insert into vector, and (d) insertion of singly restricted insert into phosphatased vector. The effects of varying vector and insert sizes, starting concentrations, and phosphatase treatment on the yield of circular 1:1 recombinants were analyzed. Selected theoretical predictions were experimentally tested and verified. Our suggestions on optimizing ligation reactions in several cases are at variance with common practice. For example, optimum conditions in case (b) and (d) ligations are best specified as individual insert and vector concentrations rather than as insert/vector molar ratios, except in case (d) ligations involving very small insert size. In case (c) ligations, highest efficiencies are obtained when both vector and insert are at relatively low concentration.