Plant DNA isolation
Recent years have seen an explosion in the number and variety of plant
molecular biology applications being used in research laboratories. The
isolation of pure nucleic acids from plant materials presents special
challenges, and commonly used molecular biology techniques often require
adaptation before they can be used with plant samples.
Many protocols have been used in plant DNA isolation, but because of the
chemical heterogeneity of the species many of them could be applied to a
limited number of species or even closely related species in some cases fail
to respond to the same protocol.
Plants, especially medicinal plants contain an array of secondary metabolites.
The compounds which make them interesting for molecular biology studies
and hence, for DNA isolation, themselves interfere with the DNA isolation
procedure. Another problem that could arise during plant DNA isolation is the
necessity of liquid nitrogen for crushing the plant material as reported in most
of the protocols and lengthy procedure involved. In many laboratories the
availability of liquid nitrogen and RNAase is a limiting factor in DNA isolation.
In order to exploit molecular cloning strategies in genetic studies it is often
necessary to compare DNA samples obtained from a large number of
individuals. Rapid screening methods have been applied to a variety of
microorganisms to follow changes in DNA sequence organization and the
insertion of foreign DNA. Isolation of plant DNA is complicated by the
presence of a tough cell wall and large amounts of polysaccharides,
phenolics, and tannins. plant extraction procedures, therefore, rely on the
isolation of nuclei and phenol extraction or preferential precipitation of DNA
followed by equilibrium density centrifugation in CsCl. Such complex and
cumbersome techniques are inappropriate for application to a large number of
samples and small amounts of tissue.
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Storage of harvested tissues:
Tissue damage can result in degradation of nucleic acids. Since tissue can
rarely be processed immediately after harvesting, storage conditions that
preserve the integrity of the nucleic acids contained in the sample are
essential. Improper storage is particularly damaging to RNA, although it can
also influence DNA quality.
When DNA is to be isolated, leaves and needles from most species can be
stored for up to 24 hours at 4°C without affecting yield or quality. In general,
samples that are to be stored for longer than 24 hours should be frozen and
kept at –80°C. However, some samples, for example, tree buds, can be
stored for several days at 4°C. Tissues stored at 4°C should be kept in a
closed container to prevent dehydration. Large samples (e.g., branches) can
be stored in a plastic bag containing a wet paper towel.
For RNA isolation, plant material should be frozen in liquid nitrogen
immediately after harvesting. Frozen samples can be stored at –80°C
indefinitely for later processing. For convenience and efficient use of space,
frozen tissue can be disrupted under liquid nitrogen and the resulting powder
stored at –80°C.
Disruption of plant material:
Complete disruption of cell walls, plasma membranes, and organelle
membranes is essential to release all the nucleic acids contained in tissue.
Insufficient disruption of starting material will lead to low yield.
Cell wall properties vary widely between different species and different
methods are required to achieve complete disruption.
Protocol for disrupting plant samples using mortar
and pestle:
The most common disruption method involves freezing samples in liquid
nitrogen and grinding with a mortar and pestle as the following:
1- Freeze tissue in liquid nitrogen immediately after harvesting.
Do not let the sample to thaw at any time during disruption.
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2. Precool mortar to –20°C and keep on dry ice.
3. Pour liquid nitrogen into the mortar, and precool pestle by placing the
grinding end in the liquid nitrogen.
4. Place frozen tissue in mortar and grind until a fine, whitish powder results.
5. Add liquid nitrogen as necessary, being careful the sample does not spill
out of the mortar.
6. Using a precooled spatula, transfer the powder to pre-cooled containers of
the appropriate size. To avoid thawing, large samples may be transferred to
several containers.
7. Ensure all liquid nitrogen has evaporated before closing the container. To
prevent the sample from thawing after evaporation, the container should be
cooled by placing it in dry ice or liquid nitrogen.
8. Proceed immediately to the DNA preparation protocol ore store in the 70°C.
DNA extraction protocol:
1- Add 1200 µl of extraction buffer (100mM Tris-HCl,pH8.0; 50mM
EDTA,PH 8.0; 500mM NaCl; 10mM ß-mercaptoethanole) to the 130mg
grounded leaves.
2- Add 100µl SDS 20% (Mix well).
3- Incubate at 65ºC for 10min (invert tubes 2-3 times.
4- Add 500µl 5M potassium acetate and place on the ice for 20 min.
5- Spine 25000 xg for 20 min at 4 ºC.
6- Pour supernatant through filter to tube containing 1ml isopropanol and
100µl ammonium acetate 5M.
7- Incubate at -20ºC for 20min.
8- Spine at 2000 xg for 20 min.
9- Wash pellet with 70% cold ethanol.
10- Spine 20000 xg for 15 min, and gently pour the supernatant and dry
pellet for 5 min.
11- Redisolve pellet with 700µl of 50Tris 10EDTA.
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12- Add 4µl RNase (100mg/ml) at 37ºC for 1 hour.
13- Add 75µl 3M sodium acetate (ph:5.2).
14- Spine 15 min to pellet insoluble debris.
15- Transfer supernatant to clean tube containing 500µl isopropanole,
allow to stand at room temperature for 5 min.
16- Pellet DNA 15 min. poure supernatant and rinse pellet with 500µl
ethanol 70%.pellet 5 min and remove ethanole.
17- Dissolve pellet in 100µl TE buffer and keep on 4ºC for 1 hour then
gently mix.
Determine the DNA integrity with agarose gel electrophoresis .
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