Should You Use Calf Intestinal Alkaline Phosphatase (CIP) in Plasmid Cloning

An obscure 1980 manuscript by Ellen Mossner and Colleagues [1] first described its isolation, but the first published description of using calf intestinal alkaline phosphatase (CIP) in gene cloning was published a while later — in Sambrook et al’s legendary 1989 manual, Molecular Cloning (2nd Edition) [2]. And so the tricky science of using CIP to dephosphorylate a vector was born. CIP works by removing the phosphate group from the 5′ end of linearised DNA, which means you can use it to dephosphorylate your vector molecule to prevent it from self-ligating and giving you the headache of a high empty vector background after ligation and transformation. But it can be a real pain to work with because: 1. It is difficult to eliminate via heat denaturation. The protocol says denature the enzyme at 65°C for 30 minutes, but this does not completely remove it. Therefore an additional clean-up step (spin column or phenol extraction) is needed. 2. It sticks like a limpet to the DNA ends, which makes it even harder to get rid of, and can interfere with ligation. 3. If residual CIP activity carries through to the ligation, it will dephosphorylate the insert, preventing ligation from occurring. 4. Anecdotally, overdoing the alkaline phosphatase treatment can damage the DNA. However, I’ve never been able to find any evidence of this or any good ideas on how it could occur, apart from in a recent discussion on the Biotechniques forum, where it was suggested that the damage might be caused by non-specific nucleases. Newer alternatives to CIP The good news is that there are a couple of new enzymes that do the job, but without so many of the drawbacks. Those are Shrimp Alkaline Phosphatase (SAP) and NEB’s Antarctic Phosphatase (AP). Both of these are more heat labile than CIP, which means they are easier to get rid of using heat denaturation. However, while even SAP is not completely eliminated by 30 min at 65°C, AP, which is totally destroyed after just 5 min at 65°C, which makes it a far superior choice. In fact, NEB say that after heat denaturation of AP treated vector, it is safe to proceed straight to the legation with no further purification step. I always add in a purification step before ligation, but I am paranoid about many things, so just ignore me. So, should you use CIP in cloning? I’d say no — why should you? In fact, why is CIP still sold as a reagent for cloning? AP is a far superior choice. Even though AP costs twice as much as CIP, I’d think you probably lose more money in failed cloning experiments using CIP, so the cheaper option is a false economy. What do you think? Do you use CIP? (Oh, and if you know whether the legend of CIP damaging DNA ends is true, and especially if you know how this happens, I’d love to know.) 1. Mossner, E., Boll, M. and Pfleiderer, G. (1980) Hoppe Seylers Z. Physiol. Chem., 361, 543-549. 2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed,), 5.72.