About DNA restriction fragment?

Question

why the DNA restriction fragments are several-fold brighter than the uncut plasmid running in a agarose gel?

Answer 1

I'm not exactly sure what you're trying to find out, but if the restriction fragments are brighter under UV light that means they are present in higher concentration than the uncut plasmid.  Band intensity is proportional to DNA concentration.

Answer 2

My guess is that you're using EtBr to stain the DNA.

In this case, remember that EtBr intercalates with the bases, this causes a conformational modification - the DNA is 'winded back' (hope I used the right word).

The restriction fragments are free at both ends and can 'uncoil' and pick up as much EtBr as it fits (I think the limit is one molecule every 4 bases or so), whereas the uncut plasmid cannot uncoil more than a certain amount without the phosphate chain breaking: for every incorporated molecule of EtBr you have an area of local 'under-coiling' that has to be compensated by another area of 'over-coiling', this area will not incorportate any EtBr. So the number of EtBr molecules it can pick up is limited because of sterical considerations.

You could check whether the 'nicked' plasmid, which has less sterical restrictions, stains more than the uncut plasmid.

Sorry, my English is particularly terrible today.