Southern blot help - (Jul/23/2007 )

I was wondering if someone could explain to me why you need to cut the genomic DNA that is electrophoresed on the gel with the same restriction enzymes that are used to generate a probe. The reason I ask is that my PI suggest I probe for an endogenous gene using the cDNA we have as a probe and cuts within the intron and exon of the gene for the digestion. Please help...

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You can cut and purify a probe template with any restriction enzyme that you choose regardless of how you chop up the genomic DNA. I could see that if the gene of interest did not have any introns, this advice would make sense but most mammalian genes do have introns. Just remember that Southerns work by hybridization of the labeled probe to the target sequence so you should be concerned about whether your gene of interest has pseudogenes or strong sequence similarity to anything else that could produce a false signal. One other caveat is to make sure the probe you cut from the cloned cDNA does not contain vector DNA. If present and labeled, this will create a lot of background and false positives. I hope this helps.

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