Isolation of wheat genomic DNA for gene mapping and cloning

Guotai Yu; Asyraf Hatta; Sambasivam Periyannan; Evans Lagudah; Brande B.H. Wulff

doi:10.1007/978-1-4939-7249-4_18

Isolation of wheat genomic DNA for gene mapping and cloning

Guotai Yu, Asyraf Hatta, Sambasivam Periyannan, Evans Lagudah, Brande B.H. Wulff^*

^*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

27 Citations (Scopus)

Abstract

DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform–isoamyl alcohol (24:1) or 6Mammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	207-213
Number of pages	7
DOIs	https://doi.org/10.1007/978-1-4939-7249-4_18
Publication status	Published - 2017

Publication series

Name	Methods in Molecular Biology
Volume	1659
ISSN (Print)	1064-3745

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10.1007/978-1-4939-7249-4_18

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title = "Isolation of wheat genomic DNA for gene mapping and cloning",

abstract = "DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform–isoamyl alcohol (24:1) or 6Mammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).",

keywords = "Cetyltrimethylammonium bromide, DNA extraction, Leaf, Seeds, Sodium dodecyl sulfate, Tissue, Wheat",

author = "Guotai Yu and Asyraf Hatta and Sambasivam Periyannan and Evans Lagudah and Wulff, {Brande B.H.}",

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AU - Hatta, Asyraf

AU - Periyannan, Sambasivam

AU - Lagudah, Evans

AU - Wulff, Brande B.H.

N1 - Publisher Copyright: © Springer Science+Business Media LLC 2017.

PY - 2017

Y1 - 2017

N2 - DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform–isoamyl alcohol (24:1) or 6Mammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).

AB - DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform–isoamyl alcohol (24:1) or 6Mammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).

KW - Cetyltrimethylammonium bromide

KW - DNA extraction

KW - Leaf

KW - Seeds

KW - Sodium dodecyl sulfate

KW - Tissue

KW - Wheat

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T3 - Methods in Molecular Biology

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BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Isolation of wheat genomic DNA for gene mapping and cloning

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