Extraction of Genomic DNA from Plant Source

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.

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

Procedure

1. Use 500 mg of young plant tissue (stem/leaf/leaf callus of controlled or transformed plant), in a precoated mortar and pestle.
2. Grind it up inside the icebox with ice, using 0.8 mL of extraction buffer and glass powder.
3. Add the rest of the buffer. Pour the ground extraction into a test tube.
4. Add 0.2 mL of 20% SDS, shake, and inoculate at 65°C–75°C for 10 to 15 minutes.
5. Add 1.5 mL of potassium acetate (5 m).
6. 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.

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.

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)

Procedure

1. Homogenize 250 mg of coconut endosperm in a mortar and pestle with lysis buffer and make up the volume to 5 mL.
2. Incubate at 50°C for 5 minutes.
3. Add 5 mL of 1:1 phenol-chloroform mixture.
4. Centrifuge for 10 minutes at 3000 rpm.
5. To the supernatant, add an equal volume of 24:1 chloroform-isopropanol mixture.
6. To the organic phase, add 120th volume of 3M sodium acetate and 2 volumes of cold absolute alcohol.
7. A white turbidity is developed.
8. 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.