The purpose of gel electrophoresis or “running a gel” is to visualize whether or not your DNA extraction and/or subsequent PCR reaction actually worked. Although PCR is supposed to only amplify a single pure product, the reality is that you end up with a mix of primer-dimers (primers binding to each other instead of the template strand) and incorrect fragments in addition to your desired product. Gel electrophoresis is used to sort DNA fragments by size (number of base pairs). By comparing PCR products to a “ladder” or a set of known standard base pair lengths, you can estimate the length of the fragments from your PCR and look for one that matches the size of the product you were trying to amplify.
What you need
To do a gel electrophoresis, you will need the following items:
- Gel rig: this is a specialized piece of equipment for casting your gel and doing an electrophoresis.
- PCR product: this is the mixture of DNA fragments you want to sort.
- DNA ladder: a solution of DNA molecules of varying length that is used as a size reference.
- Buffer: this is used to make up the gel, maintains the pH, and contains the ions necessary to carry an electric charge.
- Agarose gel: this is a type of gelatin from seaweed that will separate the DNA fragments.
- Loading dye: this dye is added to the DNA sample to make it easier to handle.
- DNA stain: this dye binds to DNA so the bands can be seen in the transilluminator. Examples include SYBR Green, SYBR Safe, Ethidium Bromide, and Fast Blast.
- Transilluminator: this machine produces UV, blue, or white light which makes the DNA stain (and the DNA) glow. Different sources of light correspond to different stains.
How does it work?
DNA molecules carry an overall negative charge. Since opposites attract, DNA is attracted to positive charges, in this case, the (+) electrode in the gel rig. To get to the (+) electrode, DNA has to travel through a sheet of agarose gel. Smaller pieces are able to travel through the gel faster than long pieces. Fragments of the same length travel at the same speed and form clear bands in the gel.
When the gel is finished running, it is soaked in a DNA Stain, a chemical that does two things: 1) bind to double-stranded DNA and 2) glow under UV, blue, or white light.
The resulting gel image should look something like this:
Note that there are 6 lanes on this gel. Lanes 1 and 6 contain a ladder that serves as a reference of measurement. The DNA or PCR product is in lanes 2-5 with resulting bands in lanes 2-4. Also note that the flow of electricity is from negative to positive.