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DNA and RNA Isolation Techniques for Non-ExpertsCTAB or SDS-Based Isolation of Plant’s DNA

DNA and RNA Isolation Techniques for Non-Experts: CTAB or SDS-Based Isolation of Plant’s DNA [Unlike the animal cell, DNA extraction from plant cells faces challenges of a rigid cellulose cell wall and the presence of a variety of cytoplasmic components like polysaccharides, polyphenols, lipids, and proteins. This demands modification in the DNA extraction protocol to obtain DNA in the purest form possible. The major modification includes the replacement of SDS with CTAB, which, apart from being a detergent, helps in the precipitation of polysaccharides under high salt concentrations. Additionally, PVP is also used during grinding as it helps in the removal of polyphenols. Both polysaccharides and polyphenols, if not removed effectively, would coprecipitate with DNA. Due to the high viscosity of CTAB, isolation buffer is maintained at 65 °C, and the homogenized tissue is also incubated at the same temperature. Post incubation in isolation buffer, DNA is extracted into the aqueous phase by chloroform-isoamyl alcohol treatment, and then it is precipitated with isopropanol. After washing this precipitated DNA in 70% ethanol, it is air-dried and resuspended in TE buffer for further use. Depending upon the cytoplasmic composition and the purpose of DNA extraction, protocols are modified for best results. Some of these modified protocols have been discussed here.] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png

DNA and RNA Isolation Techniques for Non-ExpertsCTAB or SDS-Based Isolation of Plant’s DNA

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References (7)

Publisher
Springer International Publishing
Copyright
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022
ISBN
978-3-030-94229-8
Pages
95 –105
DOI
10.1007/978-3-030-94230-4_13
Publisher site
See Chapter on Publisher Site

Abstract

[Unlike the animal cell, DNA extraction from plant cells faces challenges of a rigid cellulose cell wall and the presence of a variety of cytoplasmic components like polysaccharides, polyphenols, lipids, and proteins. This demands modification in the DNA extraction protocol to obtain DNA in the purest form possible. The major modification includes the replacement of SDS with CTAB, which, apart from being a detergent, helps in the precipitation of polysaccharides under high salt concentrations. Additionally, PVP is also used during grinding as it helps in the removal of polyphenols. Both polysaccharides and polyphenols, if not removed effectively, would coprecipitate with DNA. Due to the high viscosity of CTAB, isolation buffer is maintained at 65 °C, and the homogenized tissue is also incubated at the same temperature. Post incubation in isolation buffer, DNA is extracted into the aqueous phase by chloroform-isoamyl alcohol treatment, and then it is precipitated with isopropanol. After washing this precipitated DNA in 70% ethanol, it is air-dried and resuspended in TE buffer for further use. Depending upon the cytoplasmic composition and the purpose of DNA extraction, protocols are modified for best results. Some of these modified protocols have been discussed here.]

Published: Mar 30, 2022

Keywords: DNA extraction; Plants; Protocol; CTAB; PVP; Sequencing; High-quality DNA

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