phophorylating PCR product after CIP - (May/28/2008 )
I accidentally CIPed my DNA insert prepared by PCR. Can I rectify this by re-phosphorylating it? Or do I need to re-PCR? I needed to CIP my vector but made a mistake. I CIPed about 30-45 minutes before realizing my mistake!
If you haven't digested yet, you still can and it will leave the phosphate. If you have digested and then Cip-treated, you can rephosphorylate but you need to do a kinase reaction with T4 polynucleotide kinase. Unfortunately, this is not the most efficient reaction (not every 5' is phosphorylated) and you will need to repurify the PCR product, hence loosing some. Personally, unless you have tons of insert and all the reagents, I think you are just much better off redoing the PCR. If your ligation doesn't work you won't know where the problem was and will have to start over anyway.
When using T4 Kinase, most people do not need to add enough label for high efficiency phosphorylation. In this case, you should be adding cold ATP.
I once confirmed the data in Maniatis: at a 1:1 ratio of pmoles of label:pmoles of DNA fragment, there is 50% labeling. At 10:1, the efficiency is 90%, and at 1:10, the efficiency is 10%. 90% phosphorylation is sufficient for cloning.