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Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies

  • Genomics. Transcriptomics
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Abstract

Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful.

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Abbreviations

PC:

phenol (pH 4.5): chloroform

CIA:

chloroform: isoamyl alcohol

PCIA:

phenol: chloroform: isoamyl alcohol

CTAB:

hexadecyltrimethylammonium bromide

DTT:

dithiotreitol

PMSF:

phenylmethanesulfonyl fluoride

PVPP:

polyvinyl polypyrrolidone

DEPC:

diethyl pyrocarbonate

RH:

relative humidity

RT:

room temperature

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Authors and Affiliations

  1. Functional Genomics of Eukaryotes, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico

    A. Manoj-Kumar, K. N. Reddy & L. Blanco

  2. Department of Biotechnology, Sri Krishnadevaya University, Ananthapur, Andhra Pradesh, India

    M. Manjulatha

Authors
  1. A. Manoj-Kumar
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  2. K. N. Reddy
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  3. M. Manjulatha
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  4. L. Blanco
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Correspondence to K. N. Reddy.

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Manoj-Kumar, A., Reddy, K.N., Manjulatha, M. et al. Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies. Mol Biol 46, 535–541 (2012). https://doi.org/10.1134/S0026893312030065

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