Southern problem - No signal - Where are my bands?----southern blotting (Mar/21/2005 )

I am trying to test the knockout result by southern blotting.
I used radioactive labeling, positive charged nylon membrane and carefully followed the classical protocol. I was quite sure that the transferring was successful. But after exposure, I could see nothing in the film. I repeated twice but nothing changed. Would anyone like to tell me any possible reasons for this result and what I should do? Any comments are highly appreciated.

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hi
after transfert, did you colored with ethidium bromide membrane and gel (for see the transfert on the membrane and what remains on the gel)?

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I only did that to the membrane and saw clearly the DNA smear. Does that mean the transferring was ok? I didn't check what left on the gel.
Many thanks for your reply.

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What's the amount of the DNA that you load on the gel? Maybe you need more! I'm using 10microgram per well.

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Thank you. But I tried approximately the same amount of sample as you did.

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hi
a dna smear signify that transfert was ok. to check the efficiency better i colour the gel too.
Did you quantify the labeling of the probe?
did you run on pAcryl gel a part of your probe (i run 1µl of 30µl labelled probe)?

did you purify your probe after labelling?
i am ondering if your hybridization and or washing conditions are not too stringent for your probe...

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Hi;
What do you mean by followed the classical protocol? How long did you wash? What stingincy? Did you test yoru probe before going this? Did you test it on genomic wt DNA and check your wt band? What's halflife of your P32?

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