Agarose Gel Electrophoreses - What makes it fluoresce? - (Jan/20/2011 )

Hi there

I have always run an agarose gel in conjunction with pcr product, but my mentor said to run a gel using the plasmids that I have (without having to do pcr beforehand). I thought the primer that I was using for the pcr was important in detecting the bands, but I guess not now. This got me wondering and thinking, what makes the bands show up on the final product? I know the theory of + to -, Eth Bromide, and etc, but what makes it fluoresce? and how at just the right place (ie: why no fluorescent smearing as it goes down everytime)? My logic pattern is like when I do Western's right now. Antibody is necessary for detecting the protein, and for dna, what is necessary for detecting it?

I am just mixing my plasmid with the sample buffer and put it in the well.

If anyone can shed any light on this otherwise stupid question, I would very much appreciate it!

Thank you so much!

-Biochem_newbie-

There are different kinds of dyes that will work on DNA. I'll try to attach a picture. Ethidium bromide is an intercalating dye that binds to double stranded DNA. When it's under UV light, it fluoresces.
So any double stranded DNA will be visible on the gel. It migrates with the DNA, which is why "it's a the right place". If you run PCR product, all the DNA fragments have the same size so you will see a nice clear band on your agarose gel. If you run extract however, you'll have various sizes and you'll probably see a smear.

-Maddie-

Thanks so much for the prompt response!

So theoretically, I can just make my gel (added with Eth Bromide) as usual, and add my plasmid which includes sample buffer in the lane, and it should run properly? I don't need to add anything else to my lane?

Thanks

-Biochem_newbie-

Biochem_newbie on Thu Jan 20 16:17:35 2011 said:

Well, it's better to add a blue dye of some sort to make sure you don't run your gel too long

and a size standard of course, but that's pretty much it.

-Maddie-