never gone through such extraordinary observation, but its interesting.

Hi Gaurav,

First check whether there are any visible impurities/particles in your bottle containing old buffer. What you got is highly likely as there might be fungal growth in your old buffer. I'm assuming that you've told us about 1X TAE new and old both, because I've not seen this happen with 50X TAE stock. 

To make sure your experiment doesn't fail because of these small problems, make it a habit to always filter all buffers before use. This will always be useful in all your experiments.

Best regards with your experiments.

Its a very common mistake we do while doing the gel electrophoresis. When we use the buffer over and over again it usually gets bacterial contamination and sometimes fungal contamination too. For the degradation of plasmid also the contamination is the main cause. You can check the contamination preliminary by observing the buffer, it should be crystal clear / you can filter it to check.

One more thing all the extra (contamination) band are large in size which is clear from the marker in the 3rd well.

yeah, these bands are of genomic DNA size or larger plasmids from the buffer contaminants. I am amzed with such sharp bands without any DNA preparation.

Thanks to you all for your views.

I suspect someone used the buffer and forgot to stop the gel at the right time, and his/her DNA ran out of the gel and mixed with the buffer. If you used that same buffer, it can make its way easily into the wells.

@ Irfan- your answer seems logical but, can we get such  sharp bands without smear? Can DNA be so stable in the buffer at room temperature? The buffer was used for more than three times and stored at room temperature for more than 3-4 days.

Well, I suspect some contamination. Although your view seems logical.

Indeed yes. And you might also have seen a trail of smear behind those clear bands, suggesting DNA of different sizes present in there.