A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

Document Type : Research Articles

Authors

• Maryam M. Matin ¹
• David P. Hornby ²

¹ Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

² Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom

https://doi.org/10.22067/jcmr.v11i2.83212

Abstract

     We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Keywords

• Molecular Biology
• cDNA
• library construction
• IP RP HPLC
• size fractionation

20.1001.1.20089147.2020.11.2.2.7

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