Have you tried less DNA amount used for the digestion, or even lesser incubation time with the digestive enzyme?

Thanks for your reply Hossein, Yes I have. And my PCR products do not have smears when run on gel. I have tried with less incubation time too but it diddn't work.

it sounds like too much salt in the sample...some from pcr buffer and some from the enzyme buffer.. try running less sample or adding some water to the cut sample before running the gel. Run the gel at a lower voltage for longer time. Make sure that the running buffer in the electrophoresis tank is fresh as old buffer can have organisms growing in it that may degrade you dna product as it runs

Thanks Paul, I know it looks like one of the common problems faced during electrophoresis but trust me I have taken all the care to troubleshoot it. Infact I have also used autoclaved MQ water in Buffer of tank as well as for making gel thinking the contamination of nucleases in the buffer system, but nothing helped. Purified PCR product used with new vial of buffer and enzyme and I run the samples at 50V. Like Mathew (in between thanks for your response) I also believe its the problem of gel electrophoresis, but the PCR products are running fine. And most importantly a month back, with everything same the digestion was fine (please see the picture). Please suggest what else could go wrong, I am baffled. 

ok one difference between the product and the cut product is the cutting step. I would try incubating the product in cutting buffer without enzyme and see if there is any salt or degradation effect. Possibly also cut the product in a low salt enzyme buffer if it does cut at all in low salt. The NEB web site has percentage cutting in the wrong buffers information. I agree with Mathew that the usual reason is in the electrophoresis but all I can add to his answer is that I have seen this effect when the gel is poured too hot and the plate bends upwards so the sample comb is too close to the bottom of the gel tray. When the comb is removed the well is not sealed properly and when the sample is loaded it leaks below the gel and pools between the gel and the plate so all resolution is lost. Put some dye in a well before loading the samples and see if it spreads under the well. Try also a stronger concentration of buffer  ( the current will drop and the sample run slower but the difference between the buffer and the sam[ple salt concentration will be less).  Possibly even cut the product with another enzyme if another convenient cut site exists and run that as a positive control. Please let us know how things work

Thanks for suggestions Paul but to our disappointment I have already tried without enzyme as I mentioned earlier in my query and smearing was still there. But it doesn't occur if I incubate in water. I had tried initially with different digestion buffers but they had similar effect. And Wells are not leaky which I could see clearly. But I will try to cool the gel more than usual and see if there is any difference? I have tired with half of the concentration of digestion buffer but no change.Further I tried running the gel in 0.5X and 1X TBE buffer but still no difference. Most of the digestion buffers I am using contains BSA, do you think anything could be linked to it? The problem is...it's the same reactions I am setting which I have already done with good results a month ago. I just can't think what could go wrong in such a short span of time. It might be a very small thing which I am unable to figure out...

well done Meenu you have   covered a lot of possibilities. so this must be very frustrating.. I would phone the company that supplied the enzyme with the enzyme and buffer batch numbers    to see if they have had other complaints. I am concerned about pcr product degrading in only buffer. It suggests the buffer is highly acidic and almost all RE buffers should be around pH 8. What enzyme are you using please?.

One possibility is to check the activity of your enzyme in pcr buffer on the NEB web site. If it cuts in pcr buffer then do not bother with the RE buffer just cut the product in pcr mixture ( using more enzyme if needed). If someone in the lab has any lambda dna I might cut it with your enzyme just to make sure the company sent the correct enzyme ( very rare but possible). I general I would not inactivate the enzyme with heat particularly if it needs high temperatures which might dissociate your dna and allow the single strands to re anneal in other conformations than the original cut double stranded bands  or even denature the BSA which might bind some dna and then only release it slowly which would look like a smear. ( I have never heard of this happening and it seems unlikely as some pcr reaction have BSA or bovine thrombin added but it may be possible). If you have to inactivate  for the next step just add a little EDTA to remove the magnesium) but for the moment do not bother epecially if your samples are stored in a fridge or used immediately for the gel running.

Get another pcr product from a colleague and incubate it with your buffer to see if this unusual effect is general or specific to your sequence ( very unlikely).

If you have any other information which might help I would be very interested because, as you can see, I am struggling a bit on this problem.

As Paul mentioned, it sounds like your heat inactivation step is a probable cause. Try adding a stop buffer containing 50mM EDTA instead.

Thanks a lot Paul for your constant help to solve my query. Actually I got the answer today.. If you'll see Mathew's Answer, he mentioned to check with high melting agarose, so I tried that too. The agarose which I was using is low EEO, so I used standard agarose and got the bands right. Earlier the agarose which I was using didn't have low EEO written on the pack. Although it was the same cat# but the company had changed the product or you can say upgraded it and I started having problems. The agarose I used earlier while troubleshooting from different brands were also with low EEO. So I guess it is the agarose which created problem as Mathew earlier suggested. I still don't know why so much of difference, I think Mathew should shed some light on it. But Thanks for trying to solve my problem with me, these answers means a lot for researchers who are facing such problems. And ofcourse to Mathew too for his suggestion. 

good to hear the problem is solved Meenu and well done Mathew. Low eoo gels are always low melting/gelling point gels so possibly the gel is on the verge of melting while running so separation is poor due to diffusion of the sample in the gel

A voltage of 70 has been my preferred setting level after experiencing such. Although it increases the running time, it is better.

Best wishes Meenu