gel electrophoresis

Gel Electrophoresis

There’s a cool experiment you can do with DNA, and you only really need to know one thing: it’s a negatively charged molecule, since phosphate is negatively charged and DNA is largely made up of its sugar-phosphate backbone. The experiment is called Gel Electrophoresis, and essentially, it separates molecules based on size. Biologists use it to figure out the size of a DNA samples by seeing how far they move through a substance as compared to how far DNA fragments of known size move through the same substance.

A common substance is agarose gel, which can be easily set on a glass slide. While it’s setting, you create little wells in one end—pockets to insert your DNA into later. Usually you’ll have several samples of DNA of varying, unknown sizes combined with loading buffer, which weighs down your sample so it stays comfortably in the gel.

(Source: Wikimedia Commons)

To get started, your gel is placed into a gel electrophoresis chamber, which is essentially just a tank where you can apply a voltage and create an electric field. Running buffer is added to the tank to cover the gel, and then you insert your DNA samples into the wells. You also have a sample of DNA fragments of known size, and you usually put those on either side of your unknown samples so you can compare later.

(Source: camerazn)

When you apply a voltage across the tank, the DNA will start to move—the pores of the agarose gel act like a sieve. Since DNA is negatively charged, it will be repulsed from the negative end of the tank and migrate through the gel towards the positive end. The fragments travel parallel to each other in lanes: smaller fragments travel faster and further, and the longer fragments travel slower.

Eventually, some of the smaller molecules will have travelled all the way through the gel, and you should turn off the voltage. But at this point, you can’t clearly see where they all are, since they’re so small. So after applying a voltage, you remove the gel and stain it. Under UV light, it will be flurorescent—so voila, you can take an picture and you should see something like this:

(Source: Wikimedia Commons)

On the far left you can see the known sample, and by doing a little bit of maths and comparison, you can figure out the sizes of your DNA fragments.

Gel electrophoresis is has a bunch of applications in forensics, microbiology, genetics, biochemistry and molecular biology.

Further resources: Interactive explorations of the experiment here and here, and a more detailed look at the process from Osmania University

My lab uses EtBr for gel electrophoresis. I’ve heard that it’s more sensitive to smaller DNA bands than SYBR Safe and other alternatives and this is why its use persists. Is this really true?

Also someone in the lab seemed to think that EtBr isn’t really as bad for us as it’s been made out to be, and a couple sources I found through the wiki article on EtBr seem to corroborate this. Anyone have any input?

It’s not like I’m going to go demand that the PI start ordering SYBR dyes instead, nor am I going to go swimming in EtBr for kicks, just trying to understand the choice of EtBr here.