Making an agarose gel takes about 20 minutes from start to finish. You weigh out agarose powder, dissolve it in buffer by heating, pour it into a mold with a comb, and let it solidify at room temperature. The process is straightforward, but small details like concentration, pouring temperature, and buffer choice determine whether your gel gives you clean, well-resolved DNA bands or a blurry mess.
Choose the Right Agarose Percentage
Agarose gels work like a net: higher concentrations create a tighter mesh that slows down small DNA fragments, while lower concentrations let large fragments pass through more easily. Most gels fall between 0.5% and 2%, and the percentage you pick depends entirely on the size of DNA you’re trying to separate.
Here’s a quick guide from New England Biolabs for the best resolution at each concentration:
- 0.5% resolves fragments from 1 to 30 kb
- 0.7% resolves fragments from 0.8 to 12 kb
- 1.0% resolves fragments from 0.5 to 10 kb
- 1.2% resolves fragments from 0.4 to 7 kb
- 1.5% resolves fragments from 0.2 to 3 kb
If you’re running a standard PCR product in the 500–1,500 bp range, a 1% gel is a reliable default. For smaller fragments like restriction digests under 500 bp, go with 1.5% or even 2%. For very large genomic fragments, drop to 0.5%.
Calculate How Much Agarose to Weigh
Agarose gels are made as a weight-to-volume percentage, meaning grams of agarose per milliliters of buffer, multiplied by 100. The formula is simple: agarose in grams equals the desired percentage divided by 100, then multiplied by the volume of buffer in milliliters.
For a 1% gel using 50 mL of buffer: (1 / 100) × 50 = 0.5 g of agarose. For a 0.7% gel in 40 mL of buffer: (0.7 / 100) × 40 = 0.28 g. Weigh the agarose on an analytical balance and add it to an Erlenmeyer flask. Use a flask that holds at least three times your buffer volume, because the solution will bubble up significantly when heated.
Pick Your Buffer: TAE or TBE
The two standard buffers for agarose gel electrophoresis are TAE and TBE. Both contain Tris as a pH buffer and EDTA to protect DNA from degradation by blocking enzymes that chew up nucleic acids. The difference is the acid component: acetic acid in TAE, boric acid in TBE.
TAE gives slightly better resolution for larger DNA fragments and is the better choice if you plan to cut bands out of the gel for downstream cloning, since borate can inhibit certain enzymes. TBE produces sharper bands overall, separates smaller fragments more cleanly, and generates less electrical current (which means less heat during the run). For routine analytical gels where you just need to check fragment sizes, either works. Most teaching labs default to TAE because it’s cheaper to make in large volumes, but TBE is often preferred when resolving fragments under 1 kb.
Use the same buffer for both the gel and the running buffer in the electrophoresis tank. Mixing TAE gel with TBE running buffer (or vice versa) will ruin your results.
Melt the Agarose
Pour your measured buffer into the flask containing the agarose powder and swirl to mix. The agarose won’t dissolve yet; it needs heat. The most common method is microwaving. Heat the flask in 30-second intervals, removing it each time to swirl the contents. This prevents uneven heating and boil-overs. Repeat until the solution is completely clear with no visible particles or clumps floating around. A cloudy solution means undissolved agarose, which will create an uneven gel.
Two safety points matter here. Never seal the flask, because pressure buildup can cause it to shatter or explode. And be cautious of superheating: a liquid heated in a microwave can look calm but erupt violently when disturbed. Swirl gently after each interval and use heat-resistant gloves. You can also dissolve agarose over a Bunsen burner with constant swirling, though the microwave is faster and more commonly used.
Cool Before Pouring
This is the step most beginners rush through. Pouring agarose that’s too hot will warp or damage your gel tray. Let the flask cool on the benchtop until you can comfortably hold it for a few seconds, or place it in a 65 °C water bath. The agarose will stay liquid well above its solidification point (which is typically around 36–42 °C depending on the grade), so you have a comfortable window.
If you’re adding a DNA stain before casting, this is the time. Wait until the agarose has cooled enough that you’re not destroying the stain with excessive heat, then swirl it in gently. SYBR Safe is a common choice and only requires about 2 µL per gel. Ethidium bromide is the classic option, used at a concentration of 0.5 µg/mL, though many labs have moved away from it due to its toxicity. You can also skip pre-staining entirely and soak the gel in stain solution after electrophoresis for 15 to 30 minutes, then destain in buffer for an equal time.
Cast the Gel
Set up your gel tray in the casting apparatus. If you don’t have a casting dam, you can seal the open ends of the tray with lab tape to create a mold. Insert the comb at one end of the tray, making sure it sits just above the tray surface (not touching the bottom) so the wells will have a thin floor of agarose beneath them.
Pour the cooled agarose slowly into the mold, aiming for a steady stream to minimize air bubbles. If bubbles do appear, nudge them to the edge of the gel with a pipette tip before the gel sets. Let the gel solidify at room temperature. You’ll know it’s ready when it turns from clear to slightly opaque or translucent, which typically takes 20 to 30 minutes depending on the volume and agarose concentration.
Once set, carefully pull the comb straight up to remove it. Wiggling or angling the comb can tear the wells. Place the gel (still on its tray) into the electrophoresis box and fill the tank with running buffer until the gel is submerged by a few millimeters. Your gel is ready to load.
Storing Gels for Later Use
If you’ve cast a gel but aren’t ready to run it, wrap it in plastic wrap to prevent it from drying out and store it at 4 °C. Gels stored this way remain usable for several days, though freshly poured gels give the best results. For precast commercial gels, manufacturers recommend storing them flat at room temperature and away from light. Never freeze an agarose gel, as the ice crystals will destroy the pore structure and ruin resolution.
Common Mistakes That Ruin Gels
Using the wrong concentration is the most common reason for poor results. If your bands all cluster together at the top or bottom of the gel, your percentage doesn’t match the size range of your DNA. Incomplete dissolution of agarose creates white spots and uneven migration paths. Pouring too hot warps the tray and can create thickness gradients that make bands run crooked. Bubbles trapped under the comb produce malformed wells that leak samples into adjacent lanes.
Loading too much DNA into a well causes bands to smear rather than resolve into crisp lines. And running the gel at too high a voltage generates heat that distorts the gel matrix and produces fuzzy bands. A good rule of thumb is to run at roughly 5 to 10 volts per centimeter of distance between the electrodes. At that range, a typical mini-gel finishes in 30 to 60 minutes with clean separation.