Hi Vera, 

Don't bother to phosphorylate the dsoligo, unless you are planning on dephosphorylating your PCR product. Either way, one of these pieces of DNA needs to be phosphorylated. I tend to dephosphorylate my vector when I do cloning anyway, so in my experiments I use T4 PNK to phosphorylate the 5' end of my ordered oligos. If both pieces of DNA you are trying to ligate together are phosphorylated (or dephosphorylated), your ligation reaction will have a much lower efficiency. In my experience, T4 DNA ligase will work just fine (it's the only one I've ever used!). 

Incubating your PCR product with Taq, buffer and dNTPs I think should be okay to add an extra A at the 3' end. Unfortunately, I don't know of another enzyme which would do this job for you. An alternative would be to use Taq DNA polymerase to do the whole PCR reaction for you, then you wouldn't need to essentially do it twice (once with your proofreading DNA pol and then add the 3' A with Taq), as Taq would add the 3' A overhang anyway. Of course I'm sure you know this has the risk of incorporating mutations in your PCR product due to Taq, but it would save you a step and Taq doesn't always make mistakes. If you're planning on sequencing your ligated product afterwards, it might be worth doing the whole thing with Taq anyway. I've done this for TA cloning before, and it's worked fine. 

Good luck with your cloning! 

Hey Jonathan,

Thank you!

Just to be sure, I seem to get from your answer that my PCR product is already phosphorylated after PCR? 

Hi Vera, 

Sorry, I didn't really explain the phosphorylation issue correctly: 

If you are using 5'-phosphorylated primers to generate your PCR product, then it will automatically have the phosphate groups you require for ligation to your dsoligo (assuming that you want to add the dsoligo to the 5' end of your PCR product). If you're not using phosphorylated primers, then I would suggest that you phosphorylate them before using them in the PCR reaction (with PNK). 

If instead, you want to add your dsoligo to the 3' end of your PCR product, then you'll need to phosphorylate the dsoligo (with PNK, or order it already phosphorylated), as it won't have the free phosphate group present. In this case, I wouldn't phosphorylate the PCR primers as it's not necessary and the PCR reaction will automatically give you the 3'-hydroxyl group you need on your PCR product. 

Hi all,

whether both vector and pcr product to be phosphorylated for an increased ligation efficiency. or if both are phosphorylated what may be a possible problem?

thanking in advance

Hi Jobin Jose Kattoor,

In theory, phosphorylating both the vector and the PCR insert would provide an increased ligation efficiency, however this did not occur for me (maybe it was something to do with my sample preparation). If you are performing restriction digests upon the vector, then the sticky end will already be phosphorylated when digested, so you don't need to use a kinase to phosphorylate it. Your PCR product will not be phosphorylated automatically, unless you buy phosphorylated primers to use in the PCR reaction, or manually phosphorylate them yourself with a kinase such as PNK, or if you digest them with a restriction enzyme. If you digest them with a restriction enzyme, then the sticky end will automatically already be phosphorylated, just like what happens for a vector.

Generally, what I do is to dephosphorylate the digested vector (which helps to reduce the chance of self-ligation and therefore reduces negative background), and I don't bother using phosphorylated primers in my PCR reactions because I digest the insert anyway to ligate using sticky ends. One thing I have found helps increase my ligation efficiencies sometimes, is to perform the ligations using different molar ratios of vector to insert e.g. 1:3, 1:5, 1:10. This has helped me on some occasions when I'm having trouble getting my ligations to work.

Best wishes,

Jonathan