Running a Gel: The Actual Process
You boil agarose powder in TAE or TBE buffer, pour it into a tray with a comb, wait for it to set, pull the comb out, and drop your samples into the wells. Then you run current through it and wait. That is the whole method. Most people skip the details though, and that is where things fall apart. The concentration of agarose matters way more than people realize. A 1% gel works fine for fragments between 500 and 10,000 base pairs. If you are separating something smaller, like a 100 base pair PCR product, you need a 2% or 3% gel. If you are trying to resolve huge fragments above 15 kilobases, a regular gel is useless and you should just use pulsed-field electrophoresis instead. The principle is straightforward but the execution has nuance. DNA carries a negative charge because of its phosphate backbone. When you apply an electric field, the fragments move toward the positive electrode. Smaller fragments navigate through the gel matrix faster than larger ones. The gel acts as a molecular sieve. After the run, you stain the gel and image it under UV or blue light to see where the bands landed. The position tells you the approximate size. The intensity tells you how much DNA is there. That is the theory anyway. In practice, TAE buffer gives you better resolution for large fragments but has lower buffering capacity, so running a gel for more than 45 minutes in TAE will degrade your results as the pH shifts. TBE runs hotter and has higher capacity, which is why most protocols use it for routine work. I switched entirely to TBE after burning through half my samples running gels in TAE for extended durations. The bands just smear out because the buffer runs out.
Here is a problem I ran into last year that took me two days to figure out. I was running a restriction digest on plasmid DNA and noticed a faint smear below my expected bands that kept showing up across multiple independent preps. Turns out the sample contained trace RNA contamination from the miniprep. RNA co-migrates with small DNA fragments and ruins your interpretation. The fix was adding RNase A to the sample before loading, incubating at room temperature for ten minutes, then running the gel. I learned that the hard way after wasting three separate digest reactions trying to troubleshoot what I thought was a bad enzyme batch.
Practical Steps That Actually Work
Measure your agarose with a balance. Weighing is more accurate than guessing by volume. Mix it with your buffer in a flask, microwave it until fully dissolved, let it cool to about 60 degrees Celsius before adding any stain, then pour and let it set for 30 to 45 minutes. Preparing the samples is where most mistakes happen. Mix your DNA with loading dye. The dye has glycerol or sucrose to make the sample sink into the well, and tracking dyes like bromophenol blue to show you how far the run has progressed. Load everything carefully. A pipette tip going too deep or at too steep an angle will puncture the well and lose your sample. Run the gel at roughly 5 to 8 volts per centimeter of gel length. For a standard mini-gel that is about 10 centimeters between electrodes, that means 50 to 80 volts. Running at too high a voltage generates excess heat, melts the gel around the wells, and broadens your bands. If you are running a preparative gel where you need to extract DNA from a band afterward, use low melting point agarose and run at lower voltage for a longer time. Standard agarose gels are not suitable for extraction because the DNA gets trapped in the matrix and you lose yield. There are a few things nobody tells you about interpreting gel results. Band position is not perfectly linear with log molecular weight across the entire range. Near the bottom of the gel, where small fragments accumulate, the spacing compresses and size estimates become unreliable. If you need accurate sizing, your fragment should migrate somewhere in the middle third of the gel. Also, supercoiled plasmid runs faster than linear DNA of the same size, and nicked circular plasmid runs slower. A single plasmid prep can show up as three distinct bands, and beginners often mistake this for contamination when it is just different topological forms of the same molecule.
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Where the Method Fails
Gel electrophoresis has hard limits. Fragments larger than 30 kilobases do not separate well in standard agarose gels regardless of concentration. You need pulsed-field gel electrophoresis for that, which is a completely different setup with rotating electrodes and runs for hours or overnight. Single base pair resolution is impossible. If you need to distinguish a 500 base pair fragment from a 501 base pair fragment, a standard gel cannot do that. You would need capillary electrophoresis or sequencing instead. The method also gives you no sequence information. You know the size and approximate quantity. Nothing else. If your experiment requires knowing what the DNA actually is, this is just a screening step, not a final answer. Downloadable protocol and loading dye recipe I have compiled a one-page protocol with exact measurements for making 1%, 2%, and 3% agarose gels, sample preparation ratios, voltage settings by gel size, and a ready-to-use 6X loading dye recipe. It also includes the troubleshooting table I built from accumulated failures over the years, including the RNA contamination issue mentioned earlier. You can download it from the lab resources page. The loading dye recipe alone has saved me multiple times because I used to buy premade dye and it never lasted as long as making my own batch.
