Nucleic Acid Electrophoresis Review and Troubleshooting Guide

Nucleic acid electrophoresis is a molecular biology laboratory fundamental, used for preparing samples for downstream molecular applications, determining the results of those applications, and everything in between. The basic principle, taught in introductory courses, is simple: upon application of an electrical current, the negatively charged DNA (or RNA) is pulled through a molecular sieve, causing it to migrate toward the anode. Shorter molecules will move faster, being less retarded by the sieve, resulting in separation based on size/molecular weight.

Yet what is simple in principle may still have some practices not taught in a 101 course that it pays to know. Here we look at some tricks and tips from experts that may improve your electrophoresis experience.

First principles

The vast majority of nucleic acid analysis is run on small gels composed of polysaccharide agarose, with a higher concentration used for separation of lower molecular weight bands. These tend to be run as horizontal (“submarine”) mini-gels. Nucleic acids can also be separated in gels composed of polyacrylamide, which provides “better, higher resolution” of fragments such as smaller PCR products, notes Stephen Fratpietro, Technical Manager of the Paleo-DNA Laboratory at Lakehead University.

Once upon a time, the apparatus itself was a main trouble spot in electrophoresis. But “the rigs now are just so well made that you don’t have equipment problems anymore,” says Andor Kiss, Director, Center for Bioinformatics and Functional Genomics, and Adjunct Assistant Professor at Miami University. They tend not to leak, and the voltages are very even.

Among the biggest mistakes people make are trying to run a gel at too high a voltage or amperage, and ending up partially melting the agarose or getting “that smiley thing,” or they over- or under-load the gel, Kiss explains. He recommends starting with first principles, using the parameters recommended by the equipment manufacturer, or by a guidebook like Molecular Cloning: A Laboratory Manual—commonly known simply as Maniatis, after its original author—and adjusting from there if necessary.

Most people now run their agarose gels in tris borate EDTA buffer (TBE), he says. They used to use tris acetate EDTA (TAE) because the TBE can interfere with downstream applications such as cloning or PCR, but the cleanup and isolation kits are now so good that there is really no carryover of the TBE.

Fratpietro recommends always using fresh reagents, ensuring that the matrix is mixed evenly when making the gel, and checking liquid levels to ensure that the current has a path to travel on.

Ethidium bromide (EtBr) was once the go-to stain to visualize nucleic acid in the gel under UV light. It is still widely used, Kiss points out, but because of fears that it was toxic and mutagenic, many universities generally discourage EtBr’s use in teaching labs and require EtBr-stained gels to be treated as hazardous waste. Thus, a large number of (generally) fluorescent alternatives have been developed and are fairly widely adopted.

Beyond the mini-gel

Polyacrylamide gels—composed of polymerized acrylamide, which is neurotoxic before it is polymerized—provide a more uniform matrix, with smaller pores and greater sensitivity, capable of contending with smaller amounts of sample, than agarose. They are often purchased pre-cast, or they can be pre-made and stored in a moist, cool environment, both allowing “the gel electrophoresis to be run sooner than having to wait to make the gel first,” Fratpietro notes. He suggests making sure the sample/dye mixture falls directly to the bottom of the well to ensure an even run through the matrix.

More demanding contemporary technologies like next-generation sequencing (NGS) may require the resolution afforded by polyacrylamide, says Kiss. But “nowadays it’s probably better to run that analysis on a Bioanalyzer or Fragment Analyzer, which give both high resolution and better quantitation at the same time.”

Preparative electrophoresis

Retrieving and purifying a sample is easier from agarose than from polyacrylamide, Kiss says. Optimizing the sizes of DNA fragments for constructing NGS libraries, for example, is traditionally done by running sheared DNA on a gel and a using a razor blade to cut out a band of the appropriate size.

Researchers would eyeball where that DNA was relative to a size marker ladder. But “the size from your ladder compared to your sample don’t always line up perfectly,” points out Alex Vira, Director of Marketing at Sage Science. The other problem is that you would get a lot of cross-contamination—"DNA would actually migrate across the gel” into adjacent lanes.

Sage designed and markets a series of semi-automated solutions—including Pippin and BluePippin—that allow for isolation and capture of a particular fragment size into a small membrane-bound chamber. “Then you withdraw that from the chamber with a pipette and you're ready to go,” says Kiss. “It’s eliminated all the repetition and the loss of sample, and it has very good sample recovery. It’s pretty much the gold standard.”

Separation (and isolation) of longer fragments, such as those used by the Pacific Bio and Oxford Nanopore sequencing instruments, can benefit from a pulsed field or field inversion electrophoresis, says Vira. Here the current is periodically altered to aid the nucleic acid in snaking its way through the gel matrix.

Troubleshooting

Nucleic acid electrophoresis is generally the next workflow after conventional PCR. So if after running a gel “the gel shows no bands, faint bands, non-specific bands, primer-dimers, or smeared bands,” it might be time to revisit the PCR reaction and its protocol components and reagents, advises Oliver Glenn Hernaez, Global Product Manager, Gene Expression, Life Sciences Group at Bio-Rad Laboratories.

Although electrophoresis has become more foolproof, it always pays to consider some basic troubleshooting tips before they become necessary. For example, Hernaez advises to make sure the anode and cathode are inserted correctly, ensuring that the DNA (or RNA) is flowing from a negative to positive direction. Monitor the run so the DNA doesn’t overshoot the end of the gel. And make sure the gel has been stained so that the nucleic acid is visible (generally) under UV light.