Troubleshooting Nucleic Acid Electrophoresis

Nucleic acid electrophoresis is a multi-step process involving the preparation of gel(s), buffers, samples, and standards, plus running the gel, visualizing and documenting results, and (optionally) isolating the purified gene from the gel. Each step entails its own set of options, for example choice of buffer, elution voltage, or visualization method.

Many issues or challenges, such as faint or “smiling” bands, poor resolution, or unexpected band appearance or disappearance resolve with appropriate troubleshooting, for example by changing buffers or voltages, switching from one type of gel to another, or simply loading less or more material into the well.

“Careful sample purification and preparation is essential for gel electrophoresis. However, there are additional potential pitfalls in preparative areas for the gel itself,” says April Bauer, Ph.D., Senior Product Manager for Nucleic Acid Purification at MilliporeSigma.

For example, leak-proof assembly is essential to avoid leaking gels and simple tools can prevent user errors. Modern gel equipment employs comb structures that serve as loading guides to help identify sample wells, while color-coded lids guide correct assembly and connection to the power supply so samples run in the right direction.

Newer gel equipment setups accommodate multiple rows of samples, but to prevent errors, users must confirm the migration needed for the clear resolution of relevant bands.

It is also critical to use sufficient buffer to completely submerge the gel to ensure consistent migration of all samples throughout the depth of the gel, while commercial, ready-made buffers can eliminate errors associated with buffer prep.

“Keeping a hand on the tank and/or pipet when loading sample can provide stability, to prevent accidentally stabbing the pipet tip through the gel,” Bauer tells Biocompare. “And using a Taq or PCR master mix with a pre-mixed loading dye when analyzing PCR reactions can eliminate additional pipetting steps to prevent user error. For whatever manner is chosen, the use of loading dye mixed with the sample is critical to prevent the DNA from floating up and out of the wells and provides a visual indicator of how far the DNA has moved in the gel. This allows you to stop the gel before the DNA has run too far.”

Quality in, quality out

Unfortunately, many nucleic acid analysis problems arise not from the analysis conditions or components but the poor quality of the nucleic acid input.

For example, electrophoresis is commonly performed after polymerase chain reaction (PCR) to separate DNA fragments by length. While this application is as studied and optimized as possible it often results in the unexplained appearance or disappearance of bands on the gel.

“As a first step in troubleshooting this problem, researchers might examine their PCR reaction,” says Oliver Glenn Hernaez, Global Product Manager for Gene Expression at Bio-Rad Laboratories.

Incorrect PCR temperatures and cycling times can cause expected bands to disappear or barely appear, since too few PCR cycles can lead to insufficient target amplification.

“The general rule is that fewer cycles should be used when template concentration is high, and vice versa, with 20–35 cycles typical for a PCR run,” Hernaez says. Typical extension times are on the order of 1 minute per kilobase, but if extension time is too short target replication will similarly miss the mark.

Also, if annealing time is too short primers won’t bind to the template.

“Annealing temperature can also affect primer binding. It is recommended that the annealing temperature should be 5°C lower than the melting temperature (Tm) of the primer with the lowest Tm, but should not exceed the extension temperature.” The optimal annealing temperature for an assay can easily be determined using PCR instruments with a thermal gradient feature, such as Bio-Rad’s PTC Tempo Thermal Cyclers.

“Finally,” Hernaez explains, “if the denaturation temperature is too low or the denaturation time too short, the DNA will not denature completely, leading to low amplification. However, too high temperature or too long denaturation time can degrade the DNA. For initial denaturation, 3 min at 95°C is recommended to activate the polymerase, while 30 seconds at 95°C is suggested to denature the template during cycling.”

Sample preparation

Spurious electrophoresis results may also be a consequence of poor sample preparation, in particular during pre-electrophoresis nucleic acid purification. According to Bauer, sample quality and purity are the main challenges both before and after electrophoresis (or other downstream analysis methods).

“Choosing the right purification method to obtain high quality genomic DNA—not-sheared—is critical for downstream use in electrophoresis.”

One prep strategy involves purification through silica-based columns, but bind-wash-elute can introduce anomalous bands, process-related impurities, and degradation of products. Shearing occurs during bind-wash-elute spin preps due to multiple centrifugation steps and as a consequence of bind/release from the purification medium.

MilliporeSigma’s solution, Genelute™-E single spin nucleic acid purification kits, reduce sample handling and centrifugation steps that can damage DNA. Rather than standard silica chromatography, the kits employ negative chromatography, through which impurities are retained in the column while high molecular weight nucleic acids pass through. And since bind and wash steps are eliminated, so is that source of sample risk.

Other approaches to prevent RNA degradation include use of non-toxic stabilizers to protect samples from RNAse enzyme post purification.

Post-gel issues

“Protein, lipid, or cellular contaminants can affect downstream nucleic acid applications,” says Jeremy Lehmann, Senior Global Product Manager at Agilent Technologies. Purification helps preserve nucleic acid integrity and improve overall analysis quality and consistency.

The Agilent StrataPrep DNA Gel Extraction Kit aims to isolate pure DNA from agarose gels with minimal extraction losses by replacing resource-hogging resin manipulation, toxic phenol-chloroform extractions, and ethanol precipitations with more straightforward gel fractionation, binding to a silica-based matrix, and elution of purified DNA suitable for direct use in restriction digests, cloning, Southern blotting, PCR amplification, and other DNA analysis methods.

StrataPrep is an adaptation of a DNA purification technique based on salt precipitation, which involves digesting cellular proteins, sodium chloride “salting out” or precipitation of contaminants, DNA precipitation with ethanol, and resuspension in buffer. StrataPrep streamlines this resource-intensive set of operations with gel fractionation, silica-based binding, and simple elution, resulting in the efficient purification of DNA suitable for a variety of downstream applications.

“Throughput is limited only by centrifuge space,” Lehmann says.

According to Agilent, StrataPrep is faster than salt precipitation for extracting DNA from agarose gels, taking only two to three hours. The kit recovers up to 70–85% of DNA fragments ranging from 250 bp to 9 kb and works with DNA fragments spanning 100 bp to 23+ kb. Additionally, it is compatible with all neutral gel buffers and both conventional and low-melt agarose gels, eliminating the need for toxic reagents like phenol or chloroform.