78] Preparation of cDNA, ds cDNA, and tailed ds cDNA from interferon mRNA templates

Abstract

Publisher Summary This chapter describes specific details for the preparation of interferon cDNA for cloning. Messenger RNA can be copied into a cDNA molecule with reverse transcriptase. Interferon mRNA has been shown to be polyadenylated, which permits oligo (dT) to be used as a primer for cDNA synthesis. Complementary DNA is synthesized by the following method. Sizing of the ds cDNA allows an accurate estimation of the concentration of 3' termini. Addition of dCMP residues to the 3' ends may be followed by incorporation of [ 3 H]dCTP into acid-precipitable material. Twenty-five microcuries of [ 3 H]dCTP are used for each microgram of mRNA that is used for the original cDNA synthesis. The radioactive compound is dried and redissolved in the reaction mixture. The best transformation efficiencies with homopolymer tails of about 10-20 dCMP residues are obtained. When tails of the desired length have been generated, EDTA is added (10 mM, final concentration).