Mangesh,

How did you select for your plasmid when scaling up? Did you use the appropriate antibiotics? I've made a mistake in the past where I was working with many different kinds of vectors which required different antibiotics for selection, and while in a hurry, added the wrong antibiotic. This subsequently could lead to undesired bacterial growth, yielding a nonspecific band similar to what you described. 

Mangesh,

David's question is good. I was also wondering if you have tried to miniprep from the putatively empty DH5 prior to transformation. In other words: are you sure that the hosting bacterial strain you have used was void of plasmid? I would run a control experiment. In facts, if you have another plasmid around, that could be the reason of your results.

Another question is: why did you apparently changed the hosting strain? From your description it seems that you have done cloning in a host and then moved to DH5. I have probably misunderstood. If not why not doing maxi-prep directly from the original transformant?

Hope I have been of some help.

Luca, 

DH5 contains a recA mutation which prevents heterologous recombination, therefore increasing insert stability. That is probably why he selected the DH5 vector as opposed to his original plasmid harboring the insert. But anyways, Mangesh just downvotes peoples answers that try to help him out, so I'm not interested in helping someone who doesn't appreciate feedback from other people. Apparently he has all the answers, even though he is asking questions on this site and his experiment won't work. 

Dear David,

Thank you for your answers. I have not downvoted anyones answers. Please check again.