Authors: Sushma Tiwari* and R. S. Tomar1,
Department of Plant Breeding and Genetics, College of Agriculture, RVSKVV, Gwalior, M.P.
1National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi
*Corresponding author: firstname.lastname@example.org
The main objective of various DNA isolation methods is development of relatively quick, inexpensive and consistent protocol to extract high quality DNA with better yield. Generally leaf samples contain large quantities of polyphenols, tannins and polysaccharides. The basic principle of DNA isolation is disruption of the cell wall, cell membrane and nuclear membrane to release the highly intact DNA into solution followed by precipitation of DNA and removal of the contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols and other secondary metabolites by enzymatic or chemical methods.
Material and Methods
Leaf tissue for DNA isolation from either 7-10 day glasshouse grown seedlings or 3-4 week old plants from field.
Wheat seedlings genomic DNA isolation
DNA isolation using modified CTAB method (Murray & Thompson, 1980) for wheat is given below
- Pulverized about 2 gm of fresh young leaves in pre-chilled mortar and pestle using liquid Nitrogen.
- Transfer the powdered samples to a 50 ml polypropylene centrifuge tubes containing 10 ml of pre-warmed (650C) CTAB extraction buffer (2% w/v CTAB, 1.4M NaCl, 20mM EDTA, 100mM Tris-Cl, pH8.0) and 0.2% of β-mercaptoethanol
- Incubated samples at 650C for 1 h with occasional mixing by gentle swirling. Allow tubes to cool to room temperature.
- Emulsify the mixture of tubes with an equal volume (10 ml) of chloroform-isoamyl alcohol (24:1) for 5 min by gentle inversion and centrifuged at 15,000 rpm (Sorval RC5) for 10 min at room temperature.
- Remove the separated aqueous phase with a wide-bore pipette to a clean oak ridge tube and to it add 2/3rd volume of chilled isopropanol and mixed by quick gentle inversion for five to six times and incubated at -200C for 30 mins for coagulation of DNA.
- Spool the DNA using a bent Pasteur pipette and transferred into micro centrifuge tube and centrifuged at 10000 rpm for 5 min at room temperature
- Discard the supernatant
- Wash the DNA pellet with 70% ethanol by brief centrifugation, air dried and dissolve in TE (100 mM Tris and 50 mM EDTA, pH 8.0) buffer according to DNA pellet size and store at 40C for complete dissolution.
Major contaminants of crude DNA preparation are RNA, protein and polysaccharides. Inclusion of CTAB in DNA extraction buffer helps in elimination of polysaccharides to a large extent. The RNA is removed by treating the sample with DNase free RNase (10 mg/ml) denatured at 70 0C. Proteins including RNase can be removed by treatment with Phenol: Chloroform: Isoamylalcohol (25:24:1).
Spectrophotometric: For pure solutions of DNA, the simplest method of quantification is reading the absorbance at 260 nm where an OD of 1 in a 1 cm path length = 50 μg/ml for dsDNA, 40 μg/ml for ssRNA and 33 μg/ml for oligonucleotides and ssDNA in NanoDrop 2000 spectrophotometer (Fig. 2a & 2b). An absorbance ratio of 260 nm and 280 nm gives an estimate of the purity of the solution. Pure DNA and RNA solutions have OD 260/OD 280 values of 1.8 and 2.0, respectively (Table 2). This method is not useful for small quantities of DNA or RNA (<1 μ g/ml).DNA and RNA absorb maximally at 260 nm, proteins absorb at 280 nm and background scatter absorbs at 320 nm.
1. Murray MG and Thampson WF (1980) Rapid isolation of high molecular weight plant DNA. Nul Acids Res 8:4321-4325.
2. Singh BD and Singh AK (2016) Marker-Assisted Plant Breeding: Principles and Practices. (2015) DOI 10.1007/978-81-322-2316-0
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