Protocol

Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

As a rule of thumb, oligonucleotides >25 nucleotides should be purified by polyacrylamide gel electrophoresis, as should oligonucleotides of any length that yield anomalous results. After electrophoresis, the oligonucleotide is eluted from the gel and concentrated by reversed-phase chromatography on Sep-Pak C₁₈ columns.

The method described here is a modification of a procedure that has been in use in Michael Smith's laboratory (University of British Columbia) for more than 20 years.

MATERIALS

n-Butanol

Acetonitrile

Use 10 ml of high-performance liquid chromatography (HPLC)-grade acetonitrile for each Sep-Pak column.

Ammonium acetate (10 M)

Use 2 ml of 10 mM ammonium acetate solution . . . [Full Text of this Article]