I first tried the Roche/Boehringer Rapid Ligation Kit in the first year of my Ph.D., over 4 years ago. The kit was a free sample from a company representative when the product was first released. Since then, at least 200 constructs later, I have not ligated DNA with another product. Further, I have probably only had to repeat a handful of ligations that did not work first time. Basically, this kit almost guarentees a successful ligation.
The kit is extremely simple - dilute your DNA in a five-times dilution buffer, add an equal volume of ligation buffer, and finally 1 ul of the ligase. The buffer compositions are not revealed by Roche, but the ligation buffer is viscous and probably contains crowding agents. The only stipulation with this kit is that the buffers must be mixed extremely well before pipetting. I have always used good quality DNA for ligations, but this only constitutes using general products that are ubiquitous today (e.g. Qiagen columns and gel extraction kits). Roche suggest varying the ratio of insert to vector, but I usually find this unnecessary, just adding approximately equal amounts of both DNA. I have used the kit for ligation of sticky and blunt ends and also linkers.
Another real bonus with this product is the speed of ligation - only 5 minutes at room temperature. This means that cloning is at least a day quicker than with most other ligases. Once ligated, the DNA can be transformed straight into bacteria, or stored at -20C until transformation. I have never taken the time to see if there is a drop in efficiency as a result of storage as I have never had the need to. The efficiency of ligation is generally greater than 50% as long as the vector component of the reaction is prepared sensibly (i.e. digested, purified and redigested if the ends are not compatible and phosphatased if they are).
This kits works fantastically; the success rate is as high as the product specs suggest (for once) and there is no real time constraint imposed by using the kit. At $100 for 50 reactions, the kits also seems to make financial sense purely because the product works.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco