Northern blot DNA probes - (Aug/09/2013 )
Hello, I am making DNA probes for a Northern blot (which I have never done before) and have a question:
I am planning to make DNA probes from a PCR product, and label them with a biotin labeling kit (http://www.thermoscientificbio.com/molecular-labeling-and-detection/biotin-decalabel-dna-labeling-kit/)
Do I need to run the PCR product out on a gel first to gel purify before I label? When I do this PCR reaction it is very clean (can't see any other bands than the one I want), so it doesn't seem necessary, but most protocols I have seen include the gel purify step (unless it is to remove the taq/PCR primers/etc?)
Any other advice or tips from people who have done it this way?
You can probably label during PCR, rather than after PCR. You can spike a small amount of dUTP-biotin into a pcr reaction, which will incorporate into your product. This only works for Taq (the high fidelity enzymes will not incorporate dUTP). Much easier than a separate step after PCR, and you can control the amount of labeling. Personally, I'd choose dig-dUTP over biotin-dUTP, with the antibody from Roche. No need to buy their labeling kit -- just the dig-dUTP.
I don't think you need to purify (other than normal PCR cleanup).