Quick-Blot: Selective mRNA or DNA Immobilization from Whole Cells

Abstract

Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.