Rapid DNA Ligation Kit From Roche Applied Science

For most cloning procedures, ligation reactions are conventionally performed at 16ºC overnight; this incubation can be significantly shortened by using the Rapid DNA Ligation Kit. For simple subcloning procedures, I performed the ligation for 5 minutes at room temperature. This kit contains enough reagent for 40 ligations. The contents of the kit are ligation buffer, DNA dilution buffer and T4 DNA ligase. I have been using this kit for the past 2 years for cloning inserts of 2-4 kb into plasmid vectors.

I have compared the ligation efficiency of the Rapid DNA Ligation Kit to T4 DNA ligase from New England Biolabs (NEB) and MBI Fermentas. I set up parallel experiments to evaluate the ligation efficiencies. NEB’s ligase also allows a 5 minute ligation at room temperature and has worked the best so far in my hands. The efficiency using the same insert, vector and competent E.coli cells was 5 times better with NEB’s ligase than with the Rapid DNA Ligation Kit. The ligation efficiency with MBI’s ligase was comparable to that with the Rapid DNA Ligation Kit.

In addition to the increased ligation efficiency, there are a couple of additional advantages to the NEB enzyme. For one, the kit has an additional step of diluting the DNA in the supplied buffer whereas there is no such requirement with the NEB ligase. Also, restriction enzyme-digested DNA can be directly used (without purification) for ligation with the NEB ligase, whereas the kit requires the fragments to be gel purified. On the downside, although I am not sure about the comparative cost, NEB is certainly very expensive.

Although I had always used the ligation mix directly for transformation of competent E.coli cells, the kit recommends purifying the ligation mixture. This step takes away from the entire ‘Rapid’ DNA ligation procedure.

Overall, this kit can be economically used for fast ligation procedures on a small scale.