Agarose Gel Electrophoresis
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 spread antibiotic over-agar.
- Casting tray
- Well combs
- Voltage source
- Gel box
- UV light source
- TAE (recipe here)
- Ethidum bromide (stock concentration of 10 mg/mL)
Pouring a Standard 1% Agarose Gel:
Measure 1 g of agarose.
Mix agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe.
Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.).
Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
(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.
Pour the agarose into a gel tray with the well comb in place.
- Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.
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).
CAUTION: HOT! Be careful stirring, eruptive boiling can occur.
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.
Loading Samples and Running an Agarose Gel:
Add loading buffer to each of your DNA samples.
Once solidified, place the agarose gel into the gel box (electrophoresis unit).
Fill gel box with 1xTAE (or TBE) until the gel is covered.
Carefully load a molecular weight ladder into the first lane of the gel.
Carefully load your samples into the additional wells of the gel.
Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
(Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins.
Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
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.
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.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
Analyzing Your Gel:
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.
Purifying DNA from Your Gel:
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.
Tips and FAQ
How do you get better resolution of bands?
How do you get better separation of bands?
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.