Aptamers are high-affinity oligonucleotides which can be selected from large random libraries by systematic evolution of ligands by exponential enrichment (SELEX) protocols, with affinities and specificities comparable or better than antibodies. The SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere (with false positives and negatives) with the selection process. Here we present a primer-free protocol using a random DNA library.