Setting up a gel electrophoresis experiment is a core lab skill used to separate DNA, RNA, or proteins by size. The process involves casting an agarose gel, loading your samples into wells, and applying an electric current that pulls the molecules through the gel matrix. Smaller molecules travel faster and farther, while larger ones lag behind, creating distinct bands that you can visualize and analyze.
What Supplies Do You Need Before Starting?
Before you touch any equipment, gather everything on your bench. Running out of a reagent mid-experiment is a common and avoidable mistake.
You will need an electrophoresis chamber with a casting tray and comb, a power supply, agarose powder, and a buffer solution such as TAE or TBE. You also need your DNA samples, a loading dye, a DNA ladder or size marker, and a fluorescent stain like ethidium bromide or a safer alternative such as GelRed. Finally, have a UV or blue-light transilluminator ready for visualizing the results.
Check that your gel tray and comb are clean and free of residual agarose from previous runs. Even small debris can distort your bands.
How Do You Prepare the Agarose Gel?
The percentage of your gel determines what size fragments you can resolve. A 1% agarose gel is a good starting point for separating DNA fragments between roughly 0.5 and 5 kilobases. Use a higher percentage, such as 2%, for smaller fragments, and a lower percentage, like 0.7%, for larger ones.
Measure the agarose powder on a scale and mix it with buffer in a flask. The standard recipe is 1 gram of agarose per 100 milliliters of buffer for a 1% gel. Heat the mixture in a microwave or on a hot plate until the agarose fully dissolves. The liquid should be clear with no floating particles.
Let the molten agarose cool to about 50-60°C before pouring. If it is too hot, the gel tray may warp, and the comb can melt. If it cools too much, it will solidify unevenly. Adding your DNA stain at this stage ensures it is evenly distributed throughout the gel.
How Do You Cast the Gel Properly?
Place the casting tray in the gel rig with the rubber dams or tape firmly in place. Set the comb in position near one end of the tray. The comb creates the wells where you will load your samples.
Pour the cooled agarose into the tray in one smooth motion. Avoid creating bubbles. If bubbles appear, pop them immediately with a clean pipette tip before the gel sets. The gel should cover the comb teeth by about 2-3 millimeters.
Allow the gel to solidify at room temperature. This typically takes 20-30 minutes. You can speed this up by placing it in the refrigerator, but do not rush it to the point that the gel is brittle. The gel should feel firm and rubbery when you gently press it.
How Do You Load Samples Into the Wells?
Once the gel is solid, carefully remove the comb. Pull it straight up, not at an angle, to avoid tearing the wells. Remove the dams or tape from the tray and place the tray into the electrophoresis chamber so the wells sit at the negative (black) end.
Cover the gel with running buffer until it is submerged by about 3-5 millimeters. The buffer must completely fill the wells. If there are air bubbles trapped in the wells, dislodge them with a pipette.
Mix your DNA samples with loading dye before pipetting. The dye adds density so the sample sinks into the well, and it provides a visible tracking front as the gel runs. Use a fine pipette tip and slowly dispense the sample into the well. Do not puncture the bottom of the well with the tip. Load your DNA ladder in the first well and your samples in the remaining wells.
What Voltage Should You Run the Gel At?
Connect the chamber to the power supply. Close the lid and set the voltage. A common range is 80-120 volts for a standard gel run.
Lower voltage gives better resolution. Higher voltage runs faster but can cause the gel to heat up, which smears the bands. As a rule of thumb, run a 1% gel at no more than 5-8 volts per centimeter of gel length. For a typical 10-centimeter gel, that means 50-80 volts.
Run the gel until the loading dye has migrated about two-thirds to three-quarters of the way down the gel. This usually takes 45 minutes to 2 hours depending on the voltage and gel concentration. The tracking dye moves at a known rate, so you can judge progress visually.
How Do You Visualize and Document the Results?
Turn off the power supply and disconnect the leads before opening the lid. Remove the gel tray and carefully slide the gel onto a clean surface or directly onto the transilluminator.
If you used a stain like ethidium bromide or GelRed, the DNA bands will fluoresce under UV or blue light. Place the gel on the transilluminator and turn on the light. You should see distinct orange or green bands depending on the stain.
Capture an image immediately. DNA stains are light-sensitive, and UV exposure can damage the DNA if you plan to extract bands for further use. Wear appropriate eye and skin protection when working with UV light.
If no bands appear, check your sample concentration, the stain, and whether the current actually ran. A common beginner mistake is loading the wells with the polarity reversed, which pushes the DNA out of the gel and into the buffer.
What Are Common Mistakes and How Do You Fix Them?
Smearing is the most frequent problem. This usually comes from overloading the wells with too much DNA, running the gel at too high a voltage, or using degraded samples. Reduce the amount of DNA loaded, lower the voltage, and check the integrity of your samples on a separate gel.
Faint or missing bands often mean the stain concentration was too low or the DNA concentration was too low. Increase the stain concentration or load more sample. If your DNA is very dilute, you may need to concentrate it first.
Bands that run unevenly across the gel usually indicate a problem with the buffer. Old or contaminated buffer can create a pH gradient that distorts the run. Always use fresh buffer for best results.
If the gel melts or the buffer becomes very hot, the voltage is too high. Reduce the voltage and consider running the gel in a cold room or on ice.
Frequently Asked Questions
How long does a gel electrophoresis run take?
A typical run takes 45 minutes to 2 hours. The exact time depends on the gel percentage, the voltage, and how far the tracking dye needs to travel.
What is the difference between TAE and TBE buffer?
TAE and TBE are both common buffers, but they differ in buffering capacity. TBE holds a more stable pH during long runs, while TAE gives better resolution for larger DNA fragments.
Can I reuse the running buffer?
You can reuse it once or twice, but fresh buffer gives the most consistent results. Used buffer can have a shifted pH and reduced ionic strength, which leads to smeared bands.
Why do my DNA bands look like a smile?
This is called smiling and it happens when the gel runs too hot. The center of the gel gets hotter than the edges, causing the DNA in the middle to run faster. Lower the voltage and the problem usually resolves.
