Gel Electrophoresis of PCR Products

Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and yield. PCR products can range up to 10kb in length, but the majority of amplifications are at 1kb and below, where PAGE analysis is the most effective. In the size range from 400 to 1000 bases, the choice of native PAGE or agarose for the analysis of PCR products depends mainly upon whether the product will need to be further purified. Purification from agarose is generally more convenient. Electrophoresis reveals the size of the product band, which is compared with the predicted result. Electrophoresis also shows how much of this band was produced, and reveals the presence or absence of any unintended amplification products.

PAGE gels for PCR products formulated with the amount of cross-linker chosen to give pore sizes optimal for the size of DNA fragment desired. Gels are most often stained in ethidium bromide, even though the fluorescence of this stain is quenched by polyacrylamide, which decreases sensitivity 2-5 fold. This decrease in sensitivity generally does not present a proble, because most PCR reactions yield product levels in the microgram range, and ethidium will detect as little as one-tenth of this amount.

Interpretation


Ideally, electrophoresis yields a single strong band of correct size, as determined by comparison with size markers run on the same gel. If possible, identity should be confirmed by digestion with a restriction enzyme with a known site in the target DNA, or by Southern analysis. An unexpected band running below 100bp is usually primer-dimer. If this is the case, this band will also be found in the control reaction, run without substrate. Adjustment of Mg++ concentration may help to minimize primer-dimer, but primer redesign may be required to eliminate it completely.

Multiple bands from a PCR reaction are a bad sign. They indicate multiple priming sites for the primers within the target DNA, and call into question the reliability of the primer set. Often, multiple bands may be eliminated by raising the hybridization temperature. Sometimes adjusting the Mg++ concentration eliminates unwanted products. Reducing the primer concentration will also make the annealing more specific, eliminating incorrect amplifications. As a final resort, band(s) which appear to be correct molecular weight can be cut out and purified.

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