ISOLATION OF GENOMIC DNA FROM PLANTS
Aim
To extract and isolate genomic DNA from plant leaves.
Introduction
Isolation and purification of DNA are a crucial step in DNA molecular
techniques used in plant studies for the identification of genotypes, economical traits
associated with genes of interest, and genetic diversity. Reliable measurement of DNA
concentration and purity is also important for the assessment of food safety, especially
with the increase of the global cultivation area of genetically modified (GM) crops.
Different plant species have varying levels of polysaccharides, polyphenols, and other
secondary metabolites. These components are usually hindering the process of DNA
purification reagents
Principle
Inexpensive CTAB-based method for the extraction of high-quality genomic
DNA fro plants is adapted. Liquid nitrogen was used to break the cell wall and disrupt
the cell membrane while keeping cellular enzymes and other undesired chemicals
deactivated, thus reducing shearing and damaging of the DNA. High concentration of
CTAB was used to disrupt the cells and nuclear membranes in order to expose the
genetic components.The CTAB extraction buffer also includes NaCl which improved
the quality of the extracted DNA and 2-β-mercaptoethanol which successfully
removed polyphenols. Proteins, most lipids, and cellular debris were removed by
binding with non-aqueous compounds and precipitated during the chloroform-isoamyl
alcohol step. Longer incubation of the extracted DNA at − 20 °C also enhanced
precipitation of DNA.
Materials
3× extraction buffer containing: 3% CTAB (w/v), 1.4 M NaCl, 0.8 M Tris-HCl pH
8.0, 0.5 M EDTA pH 8.0 (autoclaved)
0.3% 2-β-Mercaptoethanol
Chloroform:isoamyl alcohol (24:1 v/v)
6 M NaCl
3 M potassium acetate
Ice cold 100% isopropyl alcohol
70% ethanol
1× TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0, autoclaved)
DNA extraction protocol
1. Preheat the 3× extraction buffer in water bath at 65 °C. Add 0.3% 2-β-
mercaptoethanol to the 3× CTAB extraction buffer immediately before use.
2. Grind 50 mg of plant samples into powder in liquid nitrogen using pre chilled
mortar and pestle. While still in the mortar, add 800 μl of the preheated 3× CTAB
extraction buffer to the grinded plant samples and swirl gently to mix using the
pestle.
3. Transfer the sample mixture to a 2-ml microcentrifuge tube, incubate in water bath
at 60–65 °C for 1 h, mix gently every 20 min by inverting the tube for 20 times
each, then cool down to the room temperature.
4. Add an equal volume of chloroform:isoamyl alcohol (24:1 v/v) and mix by slight
inversion.
5. Centrifuge at 13,000 rpm for 15 min at room temperature (RT).
6. Using a wide bore pipet, carefully transfer the upper aqueous phase, which
contains the DNA, to a new 1.5-ml eppendorf tube.
7. Repeat the extraction steps (iv–vi), when necessary until the upper aqueous phase
is clear.
8. Estimate the volume of the aqueous phase (approximately 700 μl) then add half
this volume (350 μl) of 6 M NaCl and mix well. Successively, add 1/10 the volume
(70 μl) 3 M potassium acetate and simultaneously mix with 500 μl ice cold 100%
isopropyl alcohol (approximately two thirds the volume of the aqueous phase).
Invert gently to precipitate DNA until the formation of DNA threads.
9. Incubate at − 20 °C for 30 min.
10. Centrifuge at 13,000 rpm for 5 min, discard supernatant.
11. Invert the tube containing the DNA pellet on tissue paper to complete draining off
the supernatant.
12. Wash DNA pellet with 500 μl of 70% ethanol and invert once (to dissolve residual
salts and to increase purity of the DNA).
13. Centrifuge at 13,000 rpm for 5 min.
14. Discard 70% alcohol from tubes. invert the on filter paper, and allow tubes
containing pellet to air dry at room temperature for 15 min.
15. Re-suspend the DNA pellet in 50 μl 1× TE buffer. Incubate the DNA at 50 °C for 1
to 2 h to ensure complete re-suspension.
16. Store at − 20 °C till further use.
