Agarose gel electrophoresis to assess PCR product yield: comparison with spectrophotometry, fluorometry and qPCR

Abstract

Agarose gel electrophoresis is a relatively easy to use method, commonly applied to evaluate PCR reaction success. Intercalating agents or dyes are used to visualize the amplified fragments. However, it is uncertain to what extent the brightness of bands is informative about the concentration of the amplicons. To more closely examine the suitability of agarose gel electrophoresis to assess PCR product yield, we quantified the brightness of bands on a gel and compared these data with the results from spectrophotometry, fluorometry and qPCR. Evaluation of the results suggests that assessment of the relative quantity of amplicons by band brightness is precise enough even for post-PCR analysis steps requiring PCR product concentrations within a certain range to function properly.

Keywords: PCR product quantification; agarose gel electrophoresis; fluorometry; quantitative real-time PCR; spectrophotometry.