Author:
Green Michael R.,Sambrook Joseph
Abstract
When labeled oligonucleotides are to be used in enzymatic reactions such as primer extension, virtually all of the unincorporated label must be removed from the oligonucleotide. For this purpose, chromatographic methods or gel electrophoresis are superior to differential precipitation of the oligonucleotide with ethanol or cetylpyridinium bromide (CPB). This protocol describes a method to separate labeled oligonucleotides from unincorporated label that takes advantage of differences in mobility between oligonucleotides and mononucleotides during size-exclusion chromatography. Although size-exclusion chromatography can, in principle, be used to purify either radiolabeled or nonradiolabeled oligonucleotides, this protocol is geared toward purifying radiolabeled oligonucleotides, whose elution from the column is monitored using a minimonitor and whose separation from unincorporated nucleotides is monitored by liquid scintillation counting.
Publisher
Cold Spring Harbor Laboratory
Subject
General Biochemistry, Genetics and Molecular Biology
Cited by
2 articles.
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