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Northern blot DNA probes

northern probes

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#1 Christi

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Posted 09 August 2013 - 09:04 AM

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.thermosci...a-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?



#2 phage434

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Posted 09 August 2013 - 02:28 PM

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).







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