Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).
Last Update: Feb. 20, 2018
Watch the protocol video below to learn how to perform gel electrophoresis.
Pro-Tip
Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below. Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel).
Mix agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe.
Pro-Tip
TBE can be used instead of TAE, labs usually use one or the other, but there is very little difference between the two.
Note: Make sure to use the same buffer as the one in the gel box (do not mix different buffers and do not use water).
CautionPro-Tip
It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it the solution has a tendancy to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention.
Optional: Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
Caution
EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical.
Note: If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse it in water before you can image the gel.
Pro-Tip
Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
Pro-Tip
If you are in a hurry, the gel will set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel is poured into it.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
Pro-Tip
Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has been run, just as you would if you had not added EtBr to the gel in the first place.
Carefully load a molecular weight ladder into the first lane of the gel.
Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raise the pipette straight out of the buffer.
Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.
Pro-Tip
If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes. For more details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page.
If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For instructions on how to do this, visit the Gel Purification page.
A few simple ways to increase the resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period of time; b) using a wider/thinner gel comb; or c) loading less DNA into the well. Another method for visualizing very short DNA fragments is polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments.
If you have similarly sized bands that are running too close together, you can adjust the agarose percentage of the gel to get better separation. A higher percentage agarose gel will help resolve smaller bands from each other, and a lower percentage gel will help separate larger bands.
For each sample you want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example, if you want to load 1.0 μg in 10 μL, make 1.1 μg in 11 μL.
