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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.

 

4 Comments

  1. Charles on September 19, 2016 at 6:53 pm

    Hello

    I am trying to dephosphorylate my vector PET but i am experiencing the same problem with CIAP . i am using 1 micro liter of CIAP plus 1 x ciap buffer. Gel electrophoresis shows that my Band of interest has been digested and i cannot cut it out.
    How can i solve this problem?

    I will be grateful for your advice.

    Thank you
    Charles

  2. meaningless on April 16, 2016 at 3:57 pm

    I have done a point mutation by using overlapping pcr method. I didn’t do CIP and after transformation I got over 100 colonies. I picked four of them and all of them are positive. The trick here was that after double digestion I saw three bands on the gel – parental plasmid, cut backbone and the cut insert. I gel purified the cut backbone and I think that is why I can get rid of religation of single cut parental plasmid.

    What brought me to this article was that this time I failed my site directed mutagenesis. This time the cut size was a mere 1000 and 400 and therefore I have a good chance of having both cut and uncut plasmid showing as the same band on gel. I tried to CIP it and it is now ligating. Finger cross and love to hear if I was wrong about my last cloning – that I was just damn lucky to have not picked any colonies that is transformed with the parental plasmid.

  3. NiveditaAwasthi on October 22, 2014 at 1:22 pm

    Hello, I am working with a 15kb clone, with 3 inserts. I am trying to exchange one of the inserts (~500bp) with another insert of the same size. The problem is that double digestion of vector was never 100% efficient, and each time I ligated and transformed this, I ended up with high number of background colonies. Then, I tried doing sequential digestion and used Rapid Alkaline phosphatase for dephosphorylating the vector and I saw very few colonies after transformation. The problem is that out of 8 inserts, I see good number of colonies only for 2 such inserts, which makes e doubt on everything now.

  4. ShanaSmiles on May 24, 2013 at 5:38 am

    Sigh, I use CIP because it is what my lab PI will purchase. My cloning failed recently and I thought it was due to CIP in my vector prep. So I left it out… Crazy, I know, but I got my clone and I suppose I was just lucky that pSP74 didn’t religate. My background wasn’t terribly high, but I’d like to try AP. now to convince the old timer that new, more expensive stuff, is worth a try.

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