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DNA on southern repells everything to bind - (Mar/14/2006 )


I recently faced a problem with southern blot. After hybridization, I saw no bands, but where the DNA was blotted, the membrane was brighter than the rest of the membrane (which gets slightly lila), like "white blobs". I could see the contours of the digested DNA, like on the agarose gel, as if something on the membrane (in the DNA??) had repelled anything to bind there.

I extracted the DNA with the Roche DNA isolation kit for tissues and cells and the roche DIG high prime labelling and detection starter kit I, which uses NBT/BCIP as final detection substrate.
Has anybody ever seen such a strange thing?

Thanx a lot for your help!!!


Blotting and detection are best debugged backwards. I'd recommend starting by making sure your detection works. Spot serial dilutions of your *probe* on a membrane, and get the detection working.

Then, spot serial dilutions of your genomic DNA (or other DNA to be detected) on the membrane, and make sure you can detect that.

Finally, run a gel and blot, then detect. I'd recommend, post blot, that you spot serial dilutions on the membrane as a control.

It sounds to me as if there might be too much DNA, or poor washing, or poor binding, but you need to debug the detection to figure this out.