Eliminating blurry bands in gels with a simple cost-effective repair to the gel cassette

Abstract

Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis. The appearance of blurry gel bands ofP-labeled RNA and DNA thus represents a serious problem in the laboratory. A quick search on the Internet uncovers numerous reports begrudging the appearance of blurry bands, as well as attempts to fix them without success. Indeed, our laboratories were beset by the intermittent problem of blurry gels for over one year before we found a solution. Herein we describe a simple and cost-effective solution to a problem that we show originates from the phosphorimager cassettes rather than the integrity of the gel itself. We hope that the information provided here will lead to immediate help for other laboratories experiencing similar issues with labeled nucleic acid gel-based assays. The improvement in the clarity of the gels is nothing short of astonishing in many instances and will lead to higher resolution images for analysis and publications.

<p>Variability within the human <em>TERT</em> gene, telomere length and predisposition to chronic lymphocytic leukemia</p>

“…A mutation within the splicing factor SF3B1 (splicing factor 3b subunit 1) gene in CLL was identified 45…”

“…Truncation of the protein by the introduction of premature stop codons is the most common outcome of splicing aberrations induced by SF3B1 mutations, affecting 90% of cases. In CLL, SF3B1 mutations are more frequent in later stages of the disease; they are associated with markers of poor clinical outcome and predict poor prognosis 45…”

Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17

“…Gels were dried, visualized, and quantified on a PhosphorImager using a cassette with fresh foam backing to ensure sharp bands. 46 Plots of fraction cleaved, f c , versus time were fit to either a single-exponential equation (eq 1) or a linearized version of it for very slowly reacting samples that reacted to only ∼50% completion based on the time allotted. Sample fraction cleaved versus time plots are provided in Figure S1.…”