Purification of DNA Oligos by denaturing polyacrylamide gel electrophoresis (PAGE)

Abstract

After chemical synthesis, the oligonucleotide preparation contains the desired full-length oligonucleotide but also all of the DNA molecules that were aborted during each cycle in the synthesis, and the by-products generated during the chemical reactions. The purification of oligonucleotides is a critical step for demanding applications where the exact length or sequence of the oligonucleotide is important, or for oligonucleotides longer than 50 bases. There are several methods of increasing oligonucleotide purity, the choice of which will depend on modifications of the oligonucleotides and their intended use. Polyacrylamide gel purification (PAGE purification) is the method of choice when the highest percentage of full-length oligonucleotide is desired. This chapter describes a protocol for oligonucleotide purification using denaturing polyacrylamide gel electrophoresis, and includes oligonucleotide preparation, polyacrylamide gel electrophoresis, and purification from the gel slice by two different methods: by diffusion or by electroelution. This chapter also includes recommendations as well as protocol advice.

Keywords: Butanol; Diffusion; Ethanol precipitation; Oligonucleotides; Polyacrylamide gel electrophoresis (PAGE); Preparative PAGE.