Classic Protocol in 2005

The basic strategy for enzymatic conversion of RNA into DNA has changed little since the 1970s; however, there have been great improvements to the efficiency of the overall process. In this method^1,2,3, the product of a first-strand synthesis (the cDNA-mRNA hybrid) is used as template for a nick translation reaction. Ribonuclease (RNase) H produces nicks and gaps, creating a series of RNA primers used by Escherichia coli DNA polymerase I during the synthesis of the second-strand DNA. Residual nicks are then repaired by E. coli DNA ligase, and the frayed termini of the double-stranded cDNA are polished by a DNA polymerase. Finally, bacteriophage T4 polynucleotide kinase catalyzes the phosphorylation of the ends of the cDNAs to facilitate cloning.

Two-dimensional polyacrylamide gel electrophoresis (PAGE) is used for separation of complex protein mixtures by the independent parameters of isoelectric point and molecular weight. Isoelectric focusing (IEF) separates proteins in a pH gradient. Each protein is 'focused' because it moves under the influence of the electric field until it reaches its isoelectric point, the pH at which it has no net charge. After IEF in the presence of urea and a nonionic detergent, the IEF gel is equilibrated in sodium dodecyl sulfate (SDS) to prepare the proteins for SDS-PAGE. The method described here¹ uses carrier ampholytes to form a pH gradient in a long, thin (1.2-mm) focusing gel composed of a low percentage (2.7%) of acrylamide and containing 9.5 M urea and 2% Nonidet P-40 to maintain protein solubility. After IEF, the gel is briefly equilibrated in SDS and placed directly onto the top edge of a second-dimension slab gel. Because the time between the end of IEF and the start of SDS-PAGE is only a few minutes (thereby minimizing diffusion), the spots are highly resolved and nearly round in shape.