Plant DNA isolation
AIM -: Genomic DNA can be isolate from plant
cells by disrupting the tissue and then exploiting the difference in properties
between DNA, Protein, and other constituents.
Principle
•Methods
for DNA Extraction from plant tissue can be vary depending on the
material used. Essentially , any mechanical means of breaking down the cell
wall and membranes to allow access to nuclear material, without it’s
degradation is required.
For this
usually an initial homogenization stage is employed to break down the cell wall
and allow access to DNA while harmful cellular enzyme and chemical remain
inactive.
•Phenol is
employed to denature and dissolve protein leaving nucleic acids in the aqueous
phase. Samples of DNA are then treated with phenol extracted with chloroform: Isoamyl alcohol
to precipetate.
Remaining protein and reduce the amount of dissolved phenol in aqueous phase.
• DNA must
be then Precipitate from the aqueous phase and wash thoroughly to remove
contaminating salt. The purified DNA is then resuspended and
stored in T.E buffer or FSMQ(sterile D/W).
•This
method has been shown to give intact genomic DNA from plant tissue. To check
the quality of extrated DNA,
sample is run on 0.8% agarose gel,
stained with EtBr and
visualized under UV-light
Requirement for
practical
§ 7.5 M
Ammonium acetate,
§ PVP 4%
(polyvinyl pyrrolidone) M.W 10k
§ Β-Mercaptoethanol
§
Equilibrate phenol
§
Chloroform: Isoamyl Alcohol:
(24:1)v/v
§ Phenol:
chloroform: Isomayl Alcohol-
(25:24:1)v/v
§Ethidium Bromide Dye: 10
mg/ml (Stock)
and working 0.5 µg/ml
§Extraction buffer: 1. 250mM Nacl
2. 25mM
EDTA (ph-8.0)
3. 0.5% SDS
4. 200mM Tric HCl
Protocol
§ Collect fresh leaves from plant and weight 1 to 2 gm, then wash
with tap water followed by D/W water.
§ transfer into mortar & pestle, crash homogenize thoroughly
with the extraction buffer (5ml) and beta-mercaptoethanol (50µl) in mortar.
§Incubate the sample at 65°C for 60 minute. Measure the volume
of homogenized sample, and add freshly prepaid 4% PVP.
§Add Ammonium acetate ½
of the final volume of sample and incubate in ice for 30 minutes.
§Centrifuge the homogenized sample at 10,000 RPM for 10 minute.
§Collect the supernatant in fresh sterilized tubes and add equal
volume of isopropyl alcohol or ethanol followed by incubate at -20°C for 30
minutes.
§Centrifuge at 10,000 rpm for 10 minute at 4°C
§Discard the supernatant & air dry pellet .
§ Dissolve
the pellet in TE buffer (add depending on pellet size).
§Add
20µl/ml of Rnase and incubate at 37 degree for 30 minutes.
§Add equal
volume of phenol : chloroform : isoamylalcohol (25:24:1) and gently mix during
Incubation for 2 minutes,
§Centrifuge
it 1000 rpm 4 degree 10 minute.
§Collect
supernatant and add equal volume of
chloroform : isoamylalcohol (24:1) followed by centrifugation 1000 rpm 4 degree
10 minute.
§Collect
aqueous phase (upper layer) and add equal volume of ethanol followed by
incubation in -20 degree for 15 minutes.
§Finally
load the aliquot on 0.8% Agarose gel, To check
the purity of DNA
§Again
centrifuge 1000 rpm 4 degree 15 minute. Discard supernatant and air dry pellet,
afterward dissolve the pellet in TE buffer/TAE/FSFM.
§Measure
the concentration and purity of DNA by obtaining the OD at A260/280 ratio with UV spectrophotometer. Pure DNA A260/280 ratio is 1.8, and pure RNA A260/280 ratio is 2.
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