Translation toeprinting assays using fluorescently labeled primers and capillary electrophoresis

Phillip S. Gould, Helen Bird, Andrew J. Easton

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The protocol described here is an adapted version of the toeprinting assay in which the oligonucleotide used to prime the reverse transcription step is labeled with a fluorescent dye instead of 32P. By using a fluorescent dye, the results of the assay are obtained within one hour by direct electrophoresis of the samples on an automated sequencing machine. This eliminates the need to cast and run polyacrylamide gels and to wait to expose dried gels. We show that an identical toeprint was found for the chloramphenicol acetyltransferase transcript using this nonradioactive method, which is in agreement with the previously published 32P-labeled method. Furthermore, in addition to being a faster and safer method, a larger region of sequence can be analyzed with one primer in a single experiment.

Original languageEnglish
Pages (from-to)397-400
Number of pages4
JournalBioTechniques
Volume38
Issue number3
Publication statusPublished - Mar 2005
Externally publishedYes

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
Fluorescent Dyes
Assays
Chloramphenicol O-Acetyltransferase
Transcription
Electrophoresis
Oligonucleotides
Gels
Reverse Transcription
Experiments
polyacrylamide gels

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Translation toeprinting assays using fluorescently labeled primers and capillary electrophoresis. / Gould, Phillip S.; Bird, Helen; Easton, Andrew J.

In: BioTechniques, Vol. 38, No. 3, 03.2005, p. 397-400.

Research output: Contribution to journalArticle

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