Chapter title |
Isolation of Wheat Genomic DNA for Gene Mapping and Cloning
|
---|---|
Chapter number | 18 |
Book title |
Wheat Rust Diseases
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7249-4_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7248-7, 978-1-4939-7249-4
|
Authors |
Guotai Yu, Asyraf Hatta, Sambasivam Periyannan, Evans Lagudah, Brande B. H. Wulff |
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 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+). |
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