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1. EXTRACTION OF
GENOMIC DNA FROM
PLANT SOURCE
M.Phool Badshah

2. Principle
 The major given protocol describes a rapid
method for the isolation of plant DNA
without the use of ultracentrifugation. The
DNA produced is of moderately high
molecular weight, which is suitable for
most restriction-end nucleases and
genomic blot analysis (Delta portal, et al,
1983). This method also helps to extract
DNA from small amounts of tissues and a
large number of plant samples, and is very
rapid.

3. Materials :
 Plant tissue
 Young leaves and callus of hibiscus
 Pestle and mortar
 Icebox and ice
 DNA extraction buffer
 5M potassium acetate
 3M sodium acetate buffer
 Phenol choloroform
 Isoamyl alcohol
 100% ethanol (ice cold) 70% ice cold ethanol
 Sterile eppendorf
 Microcentrifuge and glass powder

4. Procedure :
 Use 500 mg of young plant tissue (stem/leaf/leaf callus of
controlled or transformed plant), in a precoated mortar and
pestle.
 Grind it up inside the icebox with ice, using 0.8 mL of
extraction buffer and glass powder.
 Add the rest of the buffer. Pour the ground extraction into a
test tube.
 Add 0.2 mL of 20% SDS, shake, and inocubate at 65°C–
75°C for 10 to 15 minutes.
 Add 1.5 mL of potassium acetate (5M).
 Shake vigorously, and keep on ice for 20 minutes.
 Result :
 The DNA from plant cells has become extranded and clear
bands are seen when the DNA was seen on the gel.

5. Isolation of DNA From Coconut
Endosperm :
 Introduction: The distinctive property of DNA is its
behavior upon denaturation. The native form of cellular
DNA is a helical double-stranded structure. When the native
DNA is disrupted, the molecule loses its highly ordered
structure and random coils result. Significant molecular
dimensions accompany the disruption.
 Principle: DNA can be isolated from the cells by the phenol
extraction method. The cell wall is denatured and chelated
by SDS (sodium dodecyl sulfate) or SLS (sodium lauryl
sulfate). The tris acts as a buffer. The change in pH of the
medium is very low (about 0.3) because of tris. Action of
nucleases is minimized by using EDTA. Phenol-chloroform
treatment denatures DNA. Isopropanol is necessary for the
proper precipitation of DNA, which after being renatured by
sodium acetate, is precipitated by cold absolute ethanol.

6. Materials :
 Lysis Buffer: 10 mM Tris (pH 8)
5 mM EDTA
1% SDS
• Phenol
• Chloroform
• Isopropanol
• 3 M sodium acetate
• Cold absolute alcohol
• Sample (coconut endosperm)

7. Procedure :
 Homogenize 250 mg of coconut endosperm in a mortar and
pestle with lysis buffer and make up the volume to 5 mL.
 Incubate at 50°C for 5 minutes.
 Add 5 mL of 1:1 phenol-chloroform mixture.
 Centrifuge for 10 minutes at 3000 rpm.
 To the supernatant, add an equal volume of 24:1
chloroform-isopropanol mixture.
 To the organic phase, add 120th volume of 3M sodium
acetate and 2 volumes of cold absolute alcohol.
 A white turbidity is developed.
 By stirring it with a glass rod, fibrous DNA strands are
collected on the glass rod.
 Result : White DNA is observed in the form of fibers. This
is then confirmed with the help of electrophoresis. On
electrophoresis, it has produced a single band.