Oligonucleotide primers for cDNA synthesis (1 mg/ml)

Synthesis of first-strand cDNA is generally primed by random hexamers, oligo(dT)_12-18, or a mixture of the two. For directional cloning, primer-adaptors of the general structure ^5'p(dX)-(dR)-(dT)_x-OH^3' are used, where X = a clamp composed of four nucleotides (usually GAGA), R = a recognition site for a restriction enzyme, and (dT)_x = a homopolymeric run of 15 dT residues used for directional cloning.

Oligo(dT)_12-18 primers are present in the first-strand reaction mixture at a 10-fold (Mo-MLV H^-) to 40-fold (Mo-MLV) molar excess. Unlike random hexamers and primer-adaptors (please see below), the concentration of oligo(dT)_12-18 primers generally does not need to be optimized in the reaction mixture.

The concentration of random hexamers in the reaction mixture is critical. As the ratio of hexamers to mRNA increases, the average length of the cDNA decreases. It is therefore essential to optimize the ratio of random primers and mRNA template in a series of pilot reactions containing radiolabeled dCTP. The average size of the first-strand cDNA generated in each of the pilot reactions should be measured by alkaline agarose gel electrophoresis and autoradiography using DNA markers of known size. When setting up a large-scale reaction to synthesize first-strand cDNA, use the highest ratio of random primer to mRNA that generates cDNA of the maximum average length.

Primer-adaptors are generally less troublesome than random primers. However, it is nevertheless prudent to optimize the ratio of primer-adaptors and mRNA template in a series of pilot reactions containing radiolabeled dCTP. Measure the amount of cDNA synthesized in each reaction as outlined in Steps 5 and 6 of this protocol. When setting up a large-scale reaction to synthesize first-strand cDNA, use the lowest ratio of primer-adaptor to mRNA that generates the maximum yield of cDNA.

Prepare stock solutions of primers (1 mg/ml in 10 mM Tris-Cl [pH 7.4]). Store aliquots of the solutions at -70°C.