Questions about Agarose gel-electrophoresis...?

Question

In digesting a certain plasmid with restriction endonucleases, how do we explain if
1. One (biggest) fragment from the digestion has a size larger than the plasmid itself, and this fragment is outside the range of the molecular markers used for the digestion..contamination?
2. Why a lane for the uncut plasmid has more than one band?(since no enzyme is added, no digestion should have occurred and only one band is expected...)

it would be great if you can provide me the source of info..thanks~

Answer 1

Amazing question.I got the second question for my 3rd year B.Tech Bio-Tech viva-voce and I answered it!!
For the second one: Though the plasmid is uncut, some times under experimentation conditions and due to handling errors the closed circular stucture of few plasmids get distorted and open up slightly.These obviously migrate slowler than the original plasmids.Apart form this the ethidium bromide added to the gel has an effect on the plasmid.It causes the plasmid to undergo further coiling and looping. This give it a more compact structure than the original plasmid so it migrates faster.That is why it is always advisable to first carry out the electrophoreis without the dye then expose it to ethidium bromide in a gel rocker.
For the first question I must think,I will give you the answer later.
For time being you assume that it is because of contamination.

Answer 2

I'm afraid I don't know the answer to No.1. I have never seen that happen. I would guess contamination.

For No.2: The uncut plasmid is circular. Circular DNA runs in two ways - either it can be an open circular conformation or supercoiled circular conformation.

Supercoiled circular DNA runs faster than open circular DNA, producing the two bands.