Hi there,

If 3:1 and 5:1 insert:vector ratio are not working try to optimize the efficency of the digest and the purification of the products (gel purification) and first of all check the activity of the ligase.

Hi Mohammad,

Try to get a very clean band of insert to do it you always need to check on agarose gel before start ligation.

I also used to do it with 1:3 ratio, and when my insert was very clean, it worked when we had smear in background of insert on gel somehow it did not work. When I have an issue like that, I have redesigned primers for insert and solved the problem.

To me, if restriction cut worked well, ligation should not be an issue. Additionally, try to use New England Biolab enzymes.

I hope it helps. Goood luck in your cloning experiment...

Usually ratios 5:1 to 10:1 work in my hands (for PCR-amplified inserts). For inserts digested from a donor vector, te ratio is even lower (1:1 to 2:1). If you dont have good results, probably the problem is in previous digestions or in the ligation reaction itself (ligase or buffer quality). I dont think that increasing the ratio to huge values will solve that.

Hi Muhammad

i use 3:1 insert: vector ratio to get good ligation results and it worked every time. if you are not getting ligation then you should redesign the primers or try to change your restriction enzymes and use proper number of protective base pairs and as suggested by other researchers it will be of no use to increase your concentration if there is problem in your Restriction site selection.