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There is only a big square on the film.. help me -southern blotting problem- - (Sep/23/2008 )

I'm doing the southern blotting with a dig-labeled probe.

Before, I'd done the southern blotting very successfully. Thus, I thought that if i proceed it, I can get the same successful data. And I'm using the same materials and protocol except the membrane.

The brand name and catalog number of the membrane is the same with the former one. But my result is extremely different.

There is only a black big square dry.gif (the membrane) on the x-ray film.
According to my experience, I've reduced the CSPD ready to use and reduced time for exposing, but, nothing happened.

I don't know what I should do.

Please, give me any hint to solve this biggggg problem.


The simplest cause of a black square is no blocking, but I'm sure you've crossed that one off the list. Does the membrane have hot spots as well as cold spots (checked with a counter - crude, I know, but it's a start...)?