1. My overexpression plasmid (empty vector) is 8200 bp. But it is showing 4000-5000 bp always in the agarose gel, which might be for the supercoiled conformation. But this large difference is acceptable?
2. The same plasmid after digestion ( EcoRI & BamHI) becomes 12000+ bp in the agarose gel. Shouldn't it be around 8000-8200 as it becomes linear after digestion? If it becomes 12000+bp which is too big, is this acceptable? and why it becomes bigger.
The plasmid becomes bigger because the uncut version is coiled and supercoiled into a tight sphere which moves easily through the agarose due to its small diameter and the fact that many charged moirties are on the inside of the sphere. On linearisation the length becomes greater and all of the charged groups are on the outside of the molecule so there will be a size difference caused by changed electrophoretic mobility changes in the molecule. Having said that you are seeing a very large difference even allowing for agarose separation being rather poor at high molecular weights so you could try a restriction digest with an enzyme (s) that cuts many times in your plasmid and check that the sum of the smaller sizes approximately equals the expected vector size
As Paul Rutland mentions, the supercoil version will run with faster mobility than the actual size would indicate. So what are you seeing in your point #1 is not out of line with expectations.
However when you digest with restriction enzymes then you will see the true size. So it is concerning that you are using 12,000 bp. Is that the sum of different fragments or are you just linearizing the plasmid? If you can, post the picture, it might help us to troubleshoot. Sometimes you get incomplete digestions so you see more fragments than should be there so it adds up incorrectly.
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