Southern blotting is a great techinque for localization and at the same time should be done with more care considering several factors particularly if you are preparing the probes and solutions by yourself. DNA should be in good quality and see if you have positive signal in amplification. Attached are two articles that has southern pictures and the protocol followed is in Ciaffi et al 1999. See if your solutions are in right concentration and also try redesigning the probe.

It looks as if the conditions for hybridization/washing were not stringent enough. How to increase stringency depends on the particular protocol you are following. If using radiolabelled probes and 'classic' buffers, increase temperature or add formamide (or both).

Hi Rajeev,

1. Are you doing Southern assay for a single (or low) copy gene in the genome? If you are studying subject such as retrotransposons, it may be another story. I have seen signal like your photo from publications and my own experience doing retrotransposons, since they have many copies in a specific genome. But, I assume that most likely you are doing Southern for low-copy gene.

2. Protocol usually is just a general guide for you, sometimes you need to modify it to get the best results for your own project. I read the protocol you provided, it said 10 min 3 times 2xSSC. Sometimes, I use more than 20 minutes. But, I use a Geiger detector to measure the blot 'noise' (how 'hot') background of my blots after every wash, until the signal is '(pretty) weak' (need some experience).

3. As Alejandro suggested, you may want to wash them a little bit longer and with a more stringent condition. look like your blot still has lots of background. I then place the washed blots and X-ray films in-between a pair of 'Intensifying screens' inside a cassette (see pictures attached). The intensifying screen can enhance the signals. Then, I put this cassette in -80 C for 3-4 days (depends on the signal noise) before developing them.  

Hi Rajeev,

Good Southern blot result depends of many factors, in fact we can say more you are close to these factors the best result you will get in your blot:

1. Specificity of probe with the gene and probe length (best between 200 to 500 bp).

2. Restriction digestion protocol like right dilution of enzyme used for digestion of genomic DNA, some times this can also cause smear of genomic DNA, so optimization of enzyme concentration is very necessary. 

3. Percentage of agarose gel and duration of gel run before transfer (slower is run for best separation to obtain, at 21V for 6-8 hrs).

4. Temperature of hybridization some time vary from 60 to 65 degree C. The best thing we can optimize the hybridization temperature with annealing temperature of probe and genomic DNA.

5. Hybridization time and washing protocol.

Please have a recheck on the above mentioned factors, it might be helpful for you in getting a desired result.

Have you included any positive samples in your gel? 

Rajeev,

Include an additional washing step in 0.2 x SSC/0.1% SDS and see if it solves the problem.

Yes, try use 0.2XSSC /0.1% SDS, 65C. You don't need to start from the beginning. Just add the solution (I pre-warmed the solution to 65C) to the bottle with the blot. I would check every 10-15 minutes using a Geiger detector, so it won't wash away anything.

If the membrane never dried up, you can wash it straight away. Otherwise,I'd better  start over.

I'm in agreement with dr John Paul Mcnevin. Moreover, you can try to increase the melting temperature. Good luck!

Have you removed the un-incorporated hot dCTP by going through a column (by low speed centrifuge)?

If you are using P32 remember that it will decay to  sulfur, hydrolyzing the phosphodiester bond in the process. Hence, your probes will be extensively nicked (how much depends on the ratio of hot/cold dCTP, incorporation levels and storage time) after a while.

We keep the P32 at a designated area at -20 oC, not room temperature. At room temperature, it seems to decay faster.

You have to check if your labelled probe is pure enough or not.

During your blotting and transferring to membrane, did you use fresh prepared solution?

Rajeev,

I am not saying probe nicking due to P32 decay is the sole cause of what you see in that gel, but that it must be factored in. And yes, usually whenever you analyze radiolabelled probes by autoradiography you get unusual banding patterns due to a) the higher sensitivity of the technique vs. EtBr staining alone, b) nicking due to P32 decay, c) radiolysis, if the specific activity is high enough.

In the case of random priming with Klenow (vs PCR) what you usually get is a low molecular weight (in the hundred-of-bp ballpark) smear, as strand synthesis is primed from many different positions and Klenow is not that processive. The median size of the smear will progressively decrease as time goes by, and this _will_ affect hydridization (as anyone who has ever tried to use an old probe will tell you).

Now, in your specific case other things might be going on; for instance, if the amount of dCTP (cold+hot) were limiting, I would expect multiple stop sites during PCR. Would that explain your results?