Is this always the case (have you tried again?)? Maybe it was just a result of that one experiment or your phosphoimager might be dirty.
I developed last time and my photo quite nice but this is the first time I experience like this problem
Is there a chance that there are particulates or microbial contamination on one of your solutions? I've seen similar results in cases like that. The particulates stick to the surface of the membrane, bind the peroxidase conjugate, etc.
also the bands do not appear is all samples in spit of I have the same amount of DNA concentration
In my opinion you have a problem during washing... Seems in fact that there are excess reagents.
Is there a chance that your 0.4 M NaOH transfer solution has actually been prepared at a higher concentration? Concentrated alkali can damage nylon membranes and produce exactly the type of symptoms you are experiencing.
I think that it is prepared well because other people in Lab use it and got nice bands
Thanks for all, I solved the problem of black spot by increase washing and use SSC and 0.1%SDS. I got good bands in some samples and I will repeat extraction of DNA to the rest may be I do not extracted DNA well
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